defining the clinical and molecular spectrum of inherited
TRANSCRIPT
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Defining the clinical and molecular spectrum of inherited eye diseases in
community settings
Submitted by Siying Lin to the University of Exeter as a thesis for the degree of
Doctor of Philosophy in Medical Studies In June 2021
This thesis is available for Library use on the understanding that it is copyright material and that no quotation from the thesis may be published without proper acknowledgement.
I certify that all material in this thesis which is not my own work has been identified and that no material has previously been submitted and approved for the award of a degree by this or
any other University.
Signature: …………………………………………………………..
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ACKNOWLEDGEMENTS
First and foremost I would like to thank the families in this study, for their willingness
to participate in research and for generously sharing their time.
I would like to thank my supervisors Prof. Andrew Crosby and Dr. Emma Baple for the
opportunity to undertake a PhD and their expertise and supervision. I am grateful to
Dr Barry Chioza, Dr Gaurav Harlalka and Joe Leslie for their invaluable support in the
lab, and a special thanks to Dr Mark Gilchrist for his pastoral support. I have had the
opportunity to collaborate with many wonderful scientists and clinicians in the course
of my PhD, and I would like to thank them all for their contributions.
I am fortunate to have worked with colleagues who have become close friends,
including Ilaria D’Atri, Olivia Rickman, Hannah Jones and Joe Leslie; thank you for
keeping me sane. Special thanks to my good friends Annika Quinn, Roselin Charles
and Natalee James for doing the same.
To my long-suffering parents and sister Sim, thank you for your unwavering support
and encouragement, I could not have completed this journey without you. Thank you
to Charlie and Laurence for cheering me on at the finishing line.
The work in this thesis was funded by the University of Exeter Vice Chancellor
Scholarship.
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ABSTRACT
Inherited eye diseases are an important contributor to the burden of childhood
blindness globally. These conditions are often associated with significant phenotypic
and genetic heterogeneity and are extremely difficult to study in a general population
setting. This thesis details the study of inherited eye diseases in genetically isolated
populations including the North American Amish and rural Pakistani and Palestinian
communities. Here, an enrichment of disease-causing founder mutations arising from
common ancestry, characteristic marriage patterns and geographical isolation,
combined with the often large family sizes typical of families in these regions, enables
powerful genomic studies to identify pathogenic sequence variants. As well as
providing an important opportunity to learn about the genetic causes of inherited eye
diseases, these studies also provide desperately required healthcare benefits for the
families and populations involved.
Chapter 3 describes studies of oculocutaneous albinism (OCA) in 40 Amish and
Pakistani families. Results from comprehensive clinical, genomic and functional
studies, initiated by a search for the cause of OCA in a number of Amish families,
provide strong evidence for the pathogenicity of two common TYR gene variants
[p.(Ser192Tyr) and p.(Arg402Gln)] when inherited in cis. These variants were
previously considered gene polymorphisms although this has been heavily debated in
many studies, and these variants are currently variably reported and even potentially
excluded by clinical testing laboratories. The findings reported in this thesis have
important diagnostic implications by helping clarify the contribution of these variants
to the OCA phenotype, and by promoting the reporting the TYR
p.(Ser192Tyr)/p.(Arg402Gln) in cis haplotype as a pathogenic allele. This will likely
increase the molecular diagnoses in albinism patients with missing heritability by 25-
50%. This chapter also entails a comprehensive investigation involving genetic studies
alongside an exhaustive literature review of all published OCA genetic causes in
Pakistani families, including cross-referencing with established genomic databases to
evidence the likely causality of each gene variant.
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Chapter 4 entails clinical and genomic findings in four families with phenotypic features
highly suggestive of a ciliopathy disorder. Findings include identification of novel
SCAPER and BBS5 variants, enabling a more precise definition of the SCAPER
clinical phenotype. This work also consolidates an INPP5E c.1879C>T; p.(Gln627*)
variant, a likely pathogenic founder alteration present in Northern Pakistan, as a cause
of MORM syndrome.
Chapter 5 documents studies of families with rare and ultra-rare inherited ocular
diseases in Pakistani and Palestinian communities. This includes consolidating SDHD
dysfunction as a cause of mitochondrial disease through investigations of an extended
Palestinian family, facilitating a clearer delineation of the variable ocular (and non-
ocular) phenotypical features. Alongside this, the identification of novel and known
variants in ALDH1A3, FYCO1, TDRD7, CYP1B1, ATOH7, LRP5, FRMD7 and HPS1
in Pakistani communities contributes to an improved knowledge of the genetic
spectrum and frequencies of various forms of inherited eye diseases regionally.
The comprehensive OCA and BBS datasets provide notably improved knowledge, as
well as a centralised repository, of the genetic spectrum and regional frequencies of
the molecular causes of these conditions in the Amish and in Pakistan. Ultimately,
these findings will greatly facilitate the establishment of robust cost-effective accurate
diagnostic genetic testing, clinical management and counselling efforts. Together the
work of this thesis describes data of scientific importance, and highlights the immense
value of translational community research studies to deliver clinical benefits locally and
globally in the field of inherited eye diseases.
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TABLE OF CONTENTS
ACKNOWLEDGEMENTS .......................................................................................... 2
ABSTRACT ............................................................................................................... 3
TABLE OF CONTENTS ............................................................................................. 5
LIST OF FIGURES..................................................................................................... 9
LIST OF TABLES .................................................................................................... 11
ABBREVIATIONS.................................................................................................... 13
1 INTRODUCTION ................................................................................................... 16
1.1 Anatomy of the eye ....................................................................................... 16
1.2 Ocular development and malformations ..................................................... 18
1.2.1 General ocular development ...........................................................18
1.2.2 Development of the lens .................................................................22
1.2.3 Development of the cornea, iris and ciliary body ............................23
1.2.4 Development of the retina and RPE ...............................................24
1.2.5 Development of vitreous and hyaloid system ..................................28
1.3 Genetic studies in communities .................................................................. 29
1.3.1 The Amish and Anabaptist communities .........................................29
1.3.2 Inherited diseases in Pakistan ........................................................35
1.3.3 Inherited diseases in Palestine .......................................................36
1.4 Inherited eye diseases .................................................................................. 38
1.4.1 Gene therapy in inherited eye diseases ..........................................42
1.5 Disease gene and variant identification strategies .................................... 45
1.6 Project aims ................................................................................................... 46
2 MATERIALS AND METHODS .............................................................................. 48
2.1 Materials ........................................................................................................ 48
2.2 Clinical methods ........................................................................................... 48
2.2.1 Ethical approval for study................................................................48
2.2.2 Patient ascertainment and phenotyping ..........................................49
2.3 Molecular genetic methods .......................................................................... 50
2.3.1 Sample acquisition and data management .....................................50
2.3.2 DNA extraction and quantification ...................................................50
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2.3.3 Polymerase chain reaction (PCR) and dideoxy sequencing ...........53
2.3.4 Single nucleotide polymorphism (SNP) genotyping ........................58
2.3.5 Next generation sequencing (NGS) ................................................59
2.4 Literature review ........................................................................................... 62
3 STUDIES OF OCULOCUTANEOUS ALBINISM (OCA) IN COMMUNITIES ........ 63
3.1 Introduction ................................................................................................... 63
3.2 Evidence that the Ser192Tyr/Arg402Gln in cis Tyrosinase gene haplotype
is a disease-causing allele in oculocutaneous albinism type 1B (OCA1B) ... 66
3.2.1 Introduction .....................................................................................66
3.2.2 Materials and methods ...................................................................67
3.2.3 Results ............................................................................................70
3.2.4 Discussion ......................................................................................82
3.3 Genetic spectrum of OCA in Pakistan......................................................... 92
3.3.1 Introduction .....................................................................................92
3.3.2 Materials and methods ...................................................................92
3.3.3 Results: clinical and genetic findings ..............................................93
3.3.4 Discussion ....................................................................................105
3.4 Conclusions and future work ..................................................................... 111
4 STUDIES OF CILIOPATHIES IN COMMUNITIES .............................................. 112
4.1 Introduction ................................................................................................. 112
4.2 Consolidating the phenotypic features of MORM syndrome, and a review
of INPP5E-related disorders ............................................................................. 116
4.2.1 MORM syndrome ..........................................................................116
4.2.2 Materials and methods .................................................................116
4.2.3 Results: clinical and genetic findings ............................................117
4.2.4 Discussion ....................................................................................118
4.2.5 A novel BBS5 variant associated with BBS in a Pakistani family ..129
4.3 Phenotypic heterogeneity in an extended Amish family with BBS
associated with homozygosity for the common BBS1 p.(Met390Arg) variant
............................................................................................................................ 130
4.3.1 BBS ..............................................................................................130
4.3.2 Materials and methods .................................................................132
4.3.3 Results: clinical and genetic findings ............................................132
4.3.4 Discussion ....................................................................................135
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4.4 Delineating the expanding phenotype associated with SCAPER gene
mutation ............................................................................................................. 138
4.4.1 SCAPER syndrome ......................................................................138
4.4.2 Materials and methods .................................................................138
4.4.3 Results: clinical and genetic findings ............................................139
4.4.4 Discussion ....................................................................................147
4.5 Conclusions and future work ..................................................................... 151
5 IMPROVING KNOWLEDGE OF THE SPECTRUM AND CAUSES OF RARE AND
ULTRA-RARE GENETIC EYE DISEASES IN COMMUNITIES ............................. 154
5.1 Introduction ................................................................................................. 154
5.2 Consolidating biallelic SDHD variants as a cause of mitochondrial
complex II deficiency ........................................................................................ 156
5.2.1 Introduction ...................................................................................156
5.2.2 Materials and methods .................................................................157
5.2.3 Results: clinical and genetic findings ............................................157
5.2.4 Discussion ....................................................................................162
5.3 Informing clinical care through genomic studies in Pakistani families with
inherited ocular diseases ................................................................................. 165
5.3.1 Introduction ...................................................................................165
5.3.2 Materials and methods .................................................................165
5.3.3 Results: clinical and genetic findings ............................................166
5.3.4 Discussion ....................................................................................181
5.4 Conclusions and future work ..................................................................... 192
6 CONCLUDING COMMENTS .............................................................................. 195
REFERENCES ....................................................................................................... 201
APPENDIX A: NGS AND AUTOZYGOSITY MAPPING ........................................ 246
A.1 NGS ................................................................................................246
A.2 Autozygosity mapping .....................................................................247
A.3 Advances in sequencing technologies ............................................249
A.4 Glossary of terms ............................................................................251
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APPENDIX B: A NOVEL BBS5 VARIANT ASSOCIATED WITH BBS IN A
PAKISTANI FAMILY .............................................................................................. 253
B.1 Materials and methods ....................................................................253
B.2 Results: clinical and genetic findings ...............................................253
B.3 Discussion .......................................................................................255
APPENDIX C: GENETIC VARIANTS AND MAPPED LOCI ASSOCIATED WITH
NON-SYNDROMIC AND SYNDROMIC OCA IN PAKISTANI POPULATIONS .... 260
APPENDIX D: PRIMER PAIRS AND PCR CONDITIONS ..................................... 269
Table D1 Primer pairs used for sequencing the TYR coding exons and
associated intron-exon junctions ............................................................269
Table D2 Primer pairs used for sequencing the inherited eye disease gene
variants identified in the study................................................................269
APPENDIX E: PUBLICATIONS RELATING TO THIS RESEARCH ..................... 272
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LIST OF FIGURES
Figure 1.1 Anatomy of the human eye ................................................................................ 18
Figure 1.2 Development of the optic vesicles ..................................................................... 20
Figure 1.3 Development of the lens vesicle and optic cup .................................................. 21
Figure 1.4 Cell types and histologic layers in the adult human retina .................................. 26
Figure 1.5 Ancestral bottleneck leading to founder effect ................................................... 32
Figure 1.6 Genes associated with inherited retinal dystrophies .......................................... 41
Figure 2.1 Temperature gradient optimisation example ...................................................... 54
Figure 3.1 Pedigree diagrams, TYR genotype and functional data ..................................... 75
Figure 3.2 Foveal hypoplasia in individual homozygous for TYR
p.(Ser192Tyr)/p.(Arg402Gln) haplotype .............................................................................. 85
Figure 3.3 Novel missense TYR and OCA2 variants identified in this study ..................... 101
Figure 3.4 Pedigrees and genotype data for families 5 - 39 .............................................. 104
Figure 3.5 Family 40 pedigree and OCA2 genotype data ................................................. 105
Figure 3.6 Contribution of OCA genes to OCA within Pakistan and Europe ..................... 109
Figure 4.1 Ciliopathy abacus ............................................................................................ 114
Figure 4.2 Family 41 pedigree showing INPP5E c.1879C>T genotype data ..................... 119
Figure 4.3 Localisation of INPP5E disease-associated variants ....................................... 120
Figure 4.4 Geographical distribution of INPP5E disease-associated variants ................... 121
Figure 4.5 Family 43 pedigree showing BBS1 p.(Met390Arg) genotype data ................... 134
Figure 4.6 Amish (family 44) pedigree showing SCAPER c.2236dupA, p.(Ile746Asnfs*6)
genotype data and selected clinical images of affected individuals ................................... 147
Figure 5.1 Family 49 pedigree showing SDHD c.205G>A genotype data, neuroimaging and
images of affected individuals ........................................................................................... 158
Figure 5.2 Pedigrees and ALDH1A3 genotype data for families 50 - 51 ........................... 173
Figure 5.3 Pedigrees and FYCO1 genotype data for families 52 - 55 ............................... 174
Figure 5.4 Pedigrees and genotype data for families 56 - 61 ............................................ 176
Figure 5.5 Pedigrees and HPS1 genotype data for families 62 - 66 .................................. 177
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Figure 5.6 ALDH1A3 variants associated with anopthalmia and microphthalmia. ............. 183
Figure A1 Principles of autozygosity mapping .................................................................. 247
Figure B1 Family 42 pedigree showing BBS5 c.196delA genotype data .......................... 255
Figure B2 Contribution of BBS genes to BBS globally and within Pakistani families ......... 257
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LIST OF TABLES
Table 2.1 Reagents used in the study ................................................................................. 48
Table 2.2 Standard PCR reaction mixture ........................................................................... 55
Table 2.3 Touchdown PCR protocol .................................................................................... 56
Table 2.4 Criteria used for variant prioritisation ................................................................... 61
Table 3.1 Summary of clinical features observed in affected individuals in families 1 - 4 with
OCA ..................................................................................................................................... 71
Table 3.2 Prevalence of TYR p.(Ser192Tyr)/S192Y and p.(Arg402Gln)/R402Q variants in
OCA cohorts ......................................................................................................................... 79
Table 3.3 Potential contribution of TYR p.(Ser192Tyr)/p.(Arg402Gln) haplotype to molecular
diagnoses in OCA cohorts with missing heritability .............................................................. 80
Table 3.4 Review of individuals homozygous for both TYR p.(Ser192Tyr) and p.(Arg402Gln)
.............................................................................................................................................. 89
Table 3.5 TYR and OCA2 variants segregating with albinism identified in this study ......... 97
Table 3.6 Novel TYR and OCA2 variants identified in this study ....................................... 100
Table 4.1 Summary of clinical features observed in affected individuals in family 41 with
MORM syndrome and homozygous for the INPP5E p.(Gln627*) variant .......................... 117
Table 4.2 Summary of all reported disease-associated INPP5E variants ......................... 125
Table 4.3 Summary of genes associated with BBS ........................................................... 131
Table 4.4 Comparison of clinical findings of all affected individuals with biallelic pathogenic
SCAPER variants ............................................................................................................... 140
Table 4.5 Ocular findings of all affected individuals with biallelic pathogenic SCAPER
variants ............................................................................................................................... 144
Table 5.1 Clinical features of affected individuals with mitochondrial complex II deficiency
due to biallelic SDHD variants ............................................................................................ 159
Table 5.2 Variants responsible for inherited ocular diseases identified in families 50 - 66 .167
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Table 5.3 Ocular findings in affected individuals homozygous for FYCO1 c.2206C>T;
p.(Gln736*) ......................................................................................................................... 178
Table 5.4 Ocular findings in affected individuals homozygous for ATOH7 c.94delG;
p.(Ala32Profs*55) ............................................................................................................... 179
Table 5.5 Ocular findings in affected individuals homozygous for LRP5 c.1076C>G;
p.(Thr359Arg) ..................................................................................................................... 180
Table 5.6 Summary of all reported ALDH1A3 variants associated with anophthalmia and
microphthalmia.................................................................................................................... 187
Table 5.7 Summary of all reported TDRD7 variants associated with inherited ocular disease
............................................................................................................................................ 189
Table 5.8 Summary of all reported biallelic ATOH7 variants associated with inherited
developmental ocular disease ............................................................................................ 190
Table B1 Summary of clinical features observed in affected individuals in family 42 with BBS
and homozygous for the BBS5 c.196delA variant .............................................................. 254
Table B2 Summary of all reported variants associated with BBS in Pakistan ................... 258
Table C Genetic variants and mapped loci associated with non-syndromic and syndromic
OCA in Pakistani populations ............................................................................................. 260
Table D1 Primer pairs used for sequencing the TYR coding exons and associated intron-
exon junctions .................................................................................................................... 269
Table D2 Primer pairs used for sequencing the inherited eye disease gene variants identified
in the study .......................................................................................................................... 269
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ABBREVIATIONS
< Less than
> Greater than
≤ Less than or equal to
≥ Greater than or equal to
°C Degrees celsius
~ Approximately
AAV Adeno-associated viral
ADHD Attention deficit hyperactivity disorder
ANOVA Analysis of variance
ATP Adenosine triphosphate
BAM Binary Alignment/Map
BBS Bardet-Biedl syndrome
bHLH Basic helix-loop-helix
BLAT BLAST-like alignment tool
BN-PAGE Blue native polyacrylamide gel electrophoresis
bp Basepair
BWA Burrows-Wheeler Aligner
CaCl2 Calcium chloride
cDNA complementary DNA
CHARGE Coloboma, heart defects, atresia choanae, growth retardation, genital
abnormalities, ear abnormalities
CLIA Clinical Laboratory Improvement Amendments
CNS Central nervous system
CNV Copy number variant
CO2 Carbon dioxide
CRISPR Clustered Regularly Interspersed Palindromic Repeat
dATP Deoxyadenosine triphosphate
dbSNP Single Nucleotide Polymorphism Database
dCTP Deoxycytidine triphosphate
ddH2O Double-distilled water
dGTP Deoxyguanosine triphosphate
DMSO Dimethyl sulphoxide
DNA Deoxyribonucleic acid
dNTP Deoxynucleoside triphosphate
DP Read depth
dTTP Deoxythymidine triphosphate
EDTA Ethylenediaminetetraacetic acid
ER Endoplasmic reticulum
Exo I Exonuclease I
FEVR Familial exudative vitreoretinopathy
FHONDA Foveal hypoplasia, optic nerve decussation defects, and anterior
segment dysgenesis
FMS Illumina fragmentation solution
FOX Forkhead box
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g Gram
GATK Genome Analysis Toolkit
GC Guanine-cytosine
GDPR General Data Protection Regulation
g-force Relative centrifugal force
gnomAD Genome Aggregation Database
GRCh37/38 Genome Reference Consortium human genome build 37/38
HEK293F Human Embryonic Kidney 293 Freestyle
HGMD Human Gene Mutation Database
HPS Hermansky-Pudlak syndrome
HTA Human Tissue Authority
IAPB International Agency for the Prevention of Blindness
IC3D International Committee for Classification of Corneal Dystrophies
IGV Integrative Genome Viewer
Indel Insertion and deletion
iPSC Induced pluripotent stem cell
JBTS Joubert syndrome
kb Kilobase
kDa Kilodalton
KPK Khyber Pakhtunkhwa
LAB Lithium acetate borate
LB Lysogeny broth
LogMAR Logarithm of the Minimum Angle of Resolution
LRVC Low vision resource centre
MAF Minor allele frequency
Mb Megabase
MgCl2 Magnesium chloride
MIM Mendelian Inheritance in Man
min Minute
ml Millilitre
mm Millimetre
mM Millimolar
MORM Mental retardation, truncal obesity, retinal dystrophy and micropenis
MQ Mapping quality
MRI Magnetic resonance imaging
mRNA Messenger RNA
N Normality
NAD Nicotinamide adenine dinucleotide
NCBI National Center for Biotechnology Information
ng Nanogram
NGS Next generation sequencing
nm Nanometer
NNSPLICE Splice site prediction by neural network
NPHP Nephronophthisis
OCA Oculocutaneous albinism
OCT Ocular coherence tomography
OMIM Online Mendelian Inheritance in Man
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ONT Oxford Nanopore Technologies
OXPHOS Oxidative phosphorylation
PacBio Pacific Biosciences
PB1 Illumina Prepare BeadChip Buffer 1 solution
PCD Primary ciliary dyskinesia
PCR Polymerase chain reaction
PH-like Pleckstrin Homology-like
PM1 Illumina precipitation solution
PMSF Phenylmethylsulfonyl fluoride
PolyPhen-2 Polymorphism Phenotyping v2
PROVEAN Protein Variation Effect Analyser
PTC Premature termination codon
RA1 Illumina resuspension, hybridisation and wash solution
RGC Retinal ganglion cell
RNA Ribonucleic acid
RNA-seq Ribonucleic acid sequencing
RPE Retinal pigment epithelium
rpm Revolutions per minute
rSAP Shrimp alkaline phosphatase
SAM Sequence Alignment/Map
SDS-PAGE Sodium dodecyl sulphate polyacrylamide gel electrophoresis
sec Seconds
SIFT Sorting Intolerant From Tolerant
SMRT Single-molecule real-time
SNP Single Nucleotide Polymorphism
S.O.C Super Optimal broth with Catabolite repression
SSF SpliceSiteFinder-like
Ta Annealing temperature
TRID Translational read-through inducing drug
TUDCA Tauroursodeoxycholic acid
U Units
UCSC University of California, Santa Cruz
UK United Kingdom
USA United States of America
v Version
V Volt
VCF Variant Call Format
WES Whole exome sequencing
WoH Windows of Hope
WHO World Health Organisation
μl Microlitre
μM Micromolar
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1 INTRODUCTION
1.1 Anatomy of the eye
The eye is a highly specialised sensory organ which enables visual function through
photoreception and phototransduction. Light from the environment is absorbed by
specialised rod and cone photoreceptors in the retina, which converts light stimulus
into nerve action potentials. These electrical impulses are transmitted via bipolar cells
to ganglion cells, whose long axonal fibres converge to form the optic nerve, optic
chiasm and optic tract. This neural information is subsequently relayed to the visual
cortex in the brain, where it is processed and consciously appreciated as vision. Other
structures in the eye serve to support this basic physiological process, either by
helping to focus and transmit light to the retina, such as the cornea, lens, iris and ciliary
body, or to nourish and support the tissues of the eye, such as the choroid, aqueous
outflow system, and the lacrimal apparatus.
In humans, the eye is roughly spherical in shape with an approximate diameter of 23
mm. It is made up of three basic layers of tissue; the outer fibrous corneoscleral layer,
the middle uveal layer, and the inner retinal layer (Figure 1.1). The cornea and sclera
together form a tough fibrous envelope that protects and supports the ocular tissues.
The sclera is opaque and provides structural support for attachment of the extraocular
muscles, whilst anteriorly, the transparent cornea serves as the principle refracting
medium of the eye and roughly focuses an image onto the retina. The border of the
cornea and the sclera is known as the limbus. The middle layer, the uvea or uveal
tract, is composed of the choroid, the ciliary body and the iris. The choroid is a thin,
heavily pigmented, vascular connective tissue that lies between the sclera and the
retina. It nourishes and supports the outer retinal layer, and aids vision by absorption
of scattered light. Anteriorly, the choroid merges with the ciliary body, which is a
circumferential thickening of the uvea lying beneath the limbus. It consists of the ciliary
epithelium and the ciliary muscle, which are important in aqueous humour production
and accommodation respectively.
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The eye is divided into two segments by the ciliary body, zonules and lens. The smaller
anterior segment is filled with aqueous humour, which is secreted by the ciliary
epithelium, and helps maintain the intraocular pressure within the eye as well as
providing nutrition to the avascular cornea and lens. The larger posterior segment
contains the transparent viscoelastic vitreous gel that helps maintain the shape of the
eye. The heavily pigmented iris lies anterior to the ciliary body and lens, and contains
a central aperture, the pupil. The iris incompletely divides the anterior segment into
the larger anterior chamber and the smaller posterior chamber, which communicate
through the pupil. The contractile iris acts as an adjustable diaphragm by altering the
pupil size, thereby regulating the amount of light reaching the retina. The crystalline
lens is a transparent, biconvex structure that is attached to the ciliary body via radially
arranged suspensory ligaments or zonules. Changes in the tone of the ciliary muscle
alters the tension of the zonules, resulting in changes to the shape of the naturally
elastic lens. This then changes the refractive power of the lens, facilitating
accommodation by allowing the eye to focus from distant to near images.
The innermost layer of the eye is the photosensitive retina, which consists of two
primary layers, an inner neurosensory retina, and an outer retinal pigment epithelium
(RPE). The retina terminates anteriorly behind the ciliary body in a scalloped line
known as the ora serrata. The macula is an oval-shaped pigmented area near the
center of the retina, and this area is responsible for central high-acuity and colour
vision. The visual axis of the eye passes through the fovea centralis, a depression in
the center of the macula where cone photoreceptors are concentrated at the maximum
density to the exclusion of rod photoreceptors. The optic nerve is formed by
convergence of retinal ganglion cell (RGC) axons at the optic disc and exits the eye
through a sieve-like opening in the sclera known as the lamina cribrosa.
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Figure 1.1 Anatomy of the human eye
Abbreviations: AC, anterior chamber; RPE, retinal pigment epithelium. Adapted from Koeppen et al (1), created with BioRender.
1.2 Ocular development and malformations
1.2.1 General ocular development
The adult eye is a complex organ with multiple tissue components and cell types, each
with diverse yet specific functions. The development of the eye can be empirically
subdivided into three phases. The first phase involves induction through localised
signalling and regional specification to form the major morphological structures of the
eye. The second phase involves cellular differentiation and maturation of these
structures to form the functional eye. The third phase then involves the formation of
neuronal connections between the retina and the visual processing centre in the brain
(2). Normal ocular development is a coordinated multi-step process that is regulated
through a complex interaction of signalling molecules and transcription factors
encoded for by a cascade of genes that are activated in specific cell types in a specific
order. Vision and ocular development genes are highly conserved, suggesting similar
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genetic developmental programs across species. One such gene is PAX6, which
encodes a transcription factor that is a key regulator of several ocular developmental
processes (3). The amino acid sequence of the human Pax6 protein is identical to the
mouse protein (4) and shares 97% sequence identity to the zebrafish protein (5).
Mutation of the PAX6 gene in humans causes aniridia, characterised by partial or
complete absence of iris tissue (6), whilst mutations in the mouse Pax6 gene, known
as small-eye (Sey), cause iris hypoplasia and other ocular defects similar to human
aniridia (7). Developmental eye anomalies such as anophthalmia and microphthalmia
can also be caused by pathogenic mutations in a number of other ocular
developmental genes including SOX and OTX2 (8).
The developing embryo consists of three germinal layers: the ectoderm, the
mesoderm and the endoderm. The ectoderm then differentiates in the outer surface
ectoderm and the inner neuroectoderm. Developing vertebrate embryos also have a
multipotent stem cell population, the neural crest cells, which migrate and can
differentiate into diverse cell types. Ocular and orbital tissues develop from the
neuroectoderm, with contributions from the surface ectoderm, mesoderm, and
significantly from the neural crest cells. Neural crest cells also give rise to many facial
and skull structures, and therefore ocular and craniofacial anomalies are often seen in
syndromes such as Goldenhar syndrome and CHARGE syndrome that arise from
neural crest maldevelopment (9).
In humans, eye development begins during gastrulation, when the eye field, or the
eye-forming region, is induced within the anterior part of the neural plate (derived from
neuroectoderm). The eye field later splits along the midline during neurulation, and the
optic grooves develop, which later form the right and left optic vesicles (Figure 1.2). A
key inductive signal in this process is the sonic hedgehog protein encoded for by the
SHH gene, and failure of this process results in a midline defect known as
holoprosencephaly, associated with wide ocular phenotypic variability ranging from
isolated coloboma, microphthalmia, cyclopia, or complete anophthalmia (10).
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Figure 1.2 Development of the optic vesicles
(A) Transverse section through developing forebrain at day 22 showing development of the optic grooves. (B) Transverse section through developing forebrain at week 4; contact between the neuroepithelium of the optic vesicles and the surface ectoderm induces the formation of the lens placode. Adapted from Lamb (11).
The optic vesicles interact with and induce the overlying surface ectoderm to thicken
into the lens placode. The distal part of the optic vesicle then invaginates, forming a
bilayered optic cup. The inner layer of the optic cup will form the neural retina, whilst
the outer layer differentiates into the RPE (Figure 1.3). Invagination of the optic cup
occurs asymmetrically, with a groove, the optic fissure, developing from the ventral
side of the optic cup into the optic stalk. This fissure facilitates the entry of hyaloid
vessels that supply nutrients to the inner layer of the optic cup and the lens vesicle
during ocular development. Closure of this fissure occurs around week 5 of gestation,
and starts in the centre of the fissure, before “zipping up” both anteriorly and
posteriorly. The optic fissure closes by week 6, establishing the basic structure of the
eye; incomplete closure results in a coloboma, an ocular malformation that can affect
one or several structures in the eye including the iris, choroid, retina or optic disc.
Colobomas are most commonly located in the inferonasal quadrant of the eye, which
correlates with the location of optic fissure closure (12).
21
Figure 1.3 Development of the lens vesicle and optic cup
(A) Development of the optic vesicle and localised thickening of the surface ectoderm to form of the lens placode. (B) Invagination of the lens vesicle into the optic cup. (C) Lens pit deepens and detaches from the ectoderm to form a lens vesicle; invagination of the optic vesicle forms a bilayered optic cup which will eventually develop into the neural retina and retinal pigment epithelium (RPE) layers of the retina. Adapted from Erskine et al (13)
In parallel to the development of the optic cup, the lens placode invaginates into the
optic cup, forming a shallow depression known as the lens pit. The lens pit deepens,
closes anteriorly and detaches from the surface ectoderm, forming the lens vesicle
(Figure 1.3). Delayed or incomplete separation of the lens vesicle from the surface
ectoderm can result in an ocular malformation known as Peters anomaly, associated
with central or paracentral corneal opacities (leukoma), absence of the adjacent
posterior corneal stroma, corneal endothelium and descemet’s membrane, and a
22
variable degree of iris and lenticular adhesions to the posterior cornea. Most cases of
Peters anomaly are sporadic without an identified genetic cause, although some cases
have been associated with mutations in MAF, FOXC1, FOXE3, PITX2 and PAX6 (14).
As the optic vesicles extend outward, their proximal attachment to the developing
forebrain becomes the optic stalk. Growth of RGC axons into the optic stalk in week 7
leads to the formation of the optic nerve, connecting the eye with the visual centres of
the brain (12).
1.2.2 Development of the lens
The lens vesicle is a symmetrical unicellular epithelial structure that is almost spherical
in shape, and surrounded by a basal lamina layer that will eventually form the lens
capsule. Growth factors from the developing neural retina induce the posterior cells of
the lens vesicle to elongate, forming the primary lens fibres. The base of each
elongating lens cell remains anchored to the basal lamina posteriorly, whilst their
apices grow anteriorly, thereby filling the central cavity of the lens vesicle. The anterior
lens epithelial cells migrate from the central to the equatorial regions of the lens
vesicle, where they undergo mitosis and differentiate to form secondary lens fibres.
These surround the primary lens fibres in concentric layers in a process that continues
into adulthood, resulting in an ellipsoid-shaped lens. Differentiated lens fibre cells no
longer contain nuclei or mitochondria, and accumulate specialised proteins known as
crystallins, leading to lens transparency.
Lens development is a complex and delicate process and susceptible to disruption,
resulting in congenital lens opacities or cataracts. Over half of all congenital cataracts
are idiopathic (15); identified causes of congenital cataracts include genetic
aetiologies, intrauterine infections and teratogen exposure. Congenital cataracts due
to genetic aetiologies can be isolated (with autosomal dominant, autosomal recessive
and X-linked inheritance patterns described), associated with other ocular anomalies
such as aniridia, anterior segment dysgenesis, persistent foetal vasculature, and
posterior lenticonus, or associated with a number of chromosomal abnormalities,
genetic syndromes or metabolic diseases (16). Inherited congenital cataracts have
been associated with pathogenic variants in genes encoding lens proteins including
23
crystallins (CRYAA, CRYAB, CRYBA1, CRYBA2, CRYBA4, CRYBB1, CRYBB2,
CRYBB3, CRYGB, CRYGC, CRYGD and CRYGS), cytoskeletal structural proteins
(BFSP1 and BFSP2) and membrane proteins (GJA3, GJA8, MIP, LIM2, EPHA2, and
DNMBP), as well as the transcription factors FOXE3, PITX3, HSF4 and MAF (17).
1.2.3 Development of the cornea, iris and ciliary body
After separation of the lens vesicle, the surface ectoderm reforms. The underlying lens
vesicle then induces the overlying surface ectoderm to differentiate into the corneal
epithelium. Neural crest cells migrate to this region in three waves. The first wave of
cells migrate into the space between the surface ectoderm and the anterior surface of
the lens and give rise to the corneal endothelium and trabecular meshwork. A second
wave of cells then migrates between the corneal epithelium and endothelium to form
the corneal stroma. Later, a third wave of cells migrate to the space between the
corneal endothelium and the anterior edge of the optic cup, contributing to the ciliary
body and iris stroma (18). The iris and ciliary body also develop from the margins of
the optic cup under inductive influences from the developing lens, with the optic cup
margin ultimately forming the pupil margin.
FOXC1 and PITX2 are genes that encode transcription factors with critical roles in
regulating the process of neural crest cell migration in the developing eye. Both Foxc1
and Pitx2 are co-expressed in periocular mesenchyme (derived from neural crest cells
and mesoderm mesenchyme) in the developing mouse eye, and physically interact
through crucial functional domains (19). In humans, mutations in FOXC1 or PITX2
cause Axenfeld-Rieger syndrome, an autosomal dominant disorder characterised by
anterior segment dysgenesis and systemic abnormalities. Ocular features of Axenfeld-
Rieger syndrome mainly affect the iris, cornea and anterior chamber angle, structures
that are derived from the periocular mesenchyme, and include iris hypoplasia,
corectopia, polycoria, posterior embryotoxon, and iris strands connecting the
iridocorneal angle to the trabecular meshwork (20).
24
1.2.4 Development of the retina and RPE
All retinal tissues develop from the bilayered optic cup, with the neural retina
developing from the inner layer of the optic cup, whilst the outer layer forms the RPE.
As a result of the invagination process, the apical surface of the neural retina lies
adjacent to the apical surface of the RPE, with a potential space between the two
layers derived from the cavity of the optic cup; this intra-retinal space later disappears
as the two layers fuse. There are no real anatomic junctions formed between the cells
in apposition in the two layers (photoreceptors of the neural retinal and RPE cells)
(21). Therefore, whilst this interaction between the layers is crucial for the functional
maintenance of the photoreceptors, the attachment between layers remains weak, and
can be overcome by a number of mechanisms, resulting in a retinal detachment and
re-establishment of the potential intraretinal space between these two layers.
Differentiation of the retinal layers and cell types proceeds in a highly regulated
manner, starting at the posterior pole and proceeding peripherally in a concentric
manner; a gradient of retinal differentiation can therefore be seen within an individual
eye. Retinal vasculature follows the same concentric pattern of development, arising
from the hyaloid circulation at the optic disc and spreading peripherally, first reaching
the nasal periphery at 8 months gestation, whilst the temporal periphery is not
completely vascularised till shortly after birth at full term. As such, temporal retinal
vascularisation may not be fully developed in premature infants, explaining the greater
susceptibility of the temporal retina to retinopathy of prematurity, a retinal
vasoproliferative disorder affecting premature neonates (22). Congenital anomalies in
retinal vascular development are also associated with inherited ocular diseases such
as familial exudative vitreoretinopathy (FEVR), characterised by incomplete
vascularisation of the peripheral retina and poor vascular differentiation, which can
lead to complications such as the development of fibrovascular membranes resulting
in vitreoretinal traction, retinal folds and retinal detachment, and potentially profound
vision loss. Mutations in at least 10 genes have been associated with the FEVR
phenotype including NDP, FZD4, LRP5, TSPAN12, ZNF408, CTNNB1, KIF11,
RCBTB1, JAG1 and ATOH7 (23, 24); of these, NDP, FZD4, LRP5 and TSPAN12 have
been shown to play a role in the Norrin/Frizzled4 signalling pathway, suggesting a
crucial role for this pathway in retinal vascular development (23).
25
The primitive neural retina consists of two zones, an inner non-nucleated ‘marginal
zone’ and an outer nucleated ‘primitive zone’. At around week 7 of gestation, newly
formed cells from the mitotically active primitive zone migrate into the marginal zone
to form the inner neuroblastic layer; the outer nucleated zone is now known as the
outer neuroblastic layer. The inner neuroblastic layer eventually gives rise to the Müller
cells, ganglion cells and amacrine cells, whilst the outer neuroblastic layer gives rise
to bipolar cells, horizontal cells and primitive photoreceptor cells. The inner and outer
neuroblastic layers are surrounded by the internal and external limiting membranes
respectively, which form when the cells within these layers cease to proliferate and
start differentiating. Lamination of the neural retina occurs at approximately 8-12
weeks of gestation, with ganglion cells being the first cells to differentiate. Later, Müller
cells, amacrine cells, bipolar cells, horizontal cells, and photoreceptors are formed,
and their cell bodies and synaptic connections contribute to the nuclear and plexiform
layers seen in the familiar 10-layered retinal structure (Figure 1.4).
Each retinal cell type is not determined by an invariant cell lineage, instead, each
retinal progenitor cell appears to have the potential to form any of the mature retinal
neurons or glial cell types (25). This neural retinal development is driven by
overlapping cascades of genetic programs that help determine cell lineages and
commitment to specific cell fates, regulated by the temporal and spatial expression of
transcription factors in the retina encoded by three major groups of genes; the basic
helix-loop-helix (bHLH), the forkhead box (FOX) and the homeobox genes (26).
Variants in retina-specific transcription factors such as NR2E3 are associated with
primary retinal disorders (27), whilst variants in more widely expressed transcription
factors can result in systemic disorders with ocular manifestations; for example,
variants in NEUROD1, a bHLH transcription factor that functions in brain and pancreas
development, are associated with neonatal diabetes with retinal degeneration (28).
26
Figure 1.4 Cell types and histologic layers in the adult human retina
The internal limiting membrane (ILM) is the innermost boundary of the retina and is formed by the footplates of the Müller cells. The retinal nerve fibre layer (RNFL) is formed by the axons of the ganglion cells, whilst the ganglion cell layer (GCL) contains ganglion cell nuclei. The inner plexiform layer (IPL) is made up of the neuronal processes and synapses of bipolar, ganglion and amacrine cells, whilst the outer plexiform layer (OPL) is composed of the interconnections between the photoreceptor synaptic bodies and the horizontal and bipolar cells. The inner nuclear layer (INL) contains bipolar, amacrine, horizontal and Müller cell nuclei, whilst the outer nuclear layer (ONL) contains the cell bodies of the rod and cone photoreceptors. The external limiting membrane (ELM) is the outermost layer of the neurosensory retina; it is formed by the attachment sites of adjacent photoreceptors and Müller cells, and separates the photoreceptor inner segments and cell nuclei. The photoreceptor layer (PL) contains the inner and outer segments of the rod and cone photoreceptors. The retinal pigment epithelium (RPE) consists of a single layer of pigmented epithelial cells. Adapted from Koeppen et al (1), created with BioRender
Photoreceptors arise from the outermost cells of the primitive neural retina. Cone
photoreceptors develop first, followed by rod photoreceptors. The outer segments of
photoreceptors, which are actually modified ciliated processes, consist of stacks of
membranous discs containing opsin, a visual pigment that is key to initiation of the
phototransduction cascade. These outer segments are in close contact with the RPE.
Mutations in NRL and CRX, which encode transcription factors that regulate
photoreceptor differentiation during retinal development, have been shown to retinal
dystrophy phenotypes in humans (29, 30).
27
Development of the macula differs from the rest of the retina. The macula is first
evident as a localised increase in ganglion cell density temporal to the optic disc. This
is later followed by displacement of the ganglion cells and the formation of an area of
localised thinning, the foveal depression. The foveal structure is still immature at birth,
and it can take up to 15 - 45 months post-partum for the fovea to reach full histological
maturity, where the inner nuclear and ganglion cell layers have receded to the margins
of the fovea, leaving only densely packed cone photoreceptors in the foveal region
(31).
At birth, the ganglion cell layer at the fovea is still a single cell thick; in fact, full macula
development is not complete till 4 months postpartum. In foveal hypoplasia, the fovea
appears to be poorly developed or absent. This feature in isolation is rare, and is
usually described in association with other ocular disorders such as albinism, aniridia,
microphthalmia, retinopathy of prematurity, achromatopsia, optic nerve hypoplasia,
FEVR and Stickler syndrome (32, 33). Studies of inherited disease phenotypes
associated with foveal hypoplasia have identified a number of genes and molecular
pathways critical for normal foveal development, including the PAX6 transcription
factor and members of the Wnt signaling pathway (FZD4 and NDP) (34).
The RPE develops from the outer layer of the optic cup, and is initially comprised of a
single layer of ciliated pseudostratified columnar epithelial cells; the cilia later
disappear as melanogenesis commences. The basement membrane of the RPE forms
one of the five layers of the Bruch’s membrane, and tight junctions between the retinal
pigment epithelial cells form the outer blood-retinal barrier.
28
1.2.5 Development of vitreous and hyaloid system
The vitreous develops in the space between the inner layer of the optic cup and the
lens vesicle in three distinct stages. At 5 weeks gestation, the lentoretinal space is
occupied by the primary vitreous. This is derived from mesenchymal cells that have
entered the optic cup via the optic fissure, as well as from lens surface ectoderm cells
and neuroectoderm cells from the inner layer of the optic cup (35). The hyaloid artery,
which is a branch of the primitive ophthalmic artery, enters the optic cup via the optic
fissure and courses through the primary vitreous to reach the posterior lens surface,
where it branches to form a network of capillaries, the tunica vasculosa lentis.
The secondary vitreous is derived entirely from the neuroectoderm, and is composed
of fine, organised fibrillary material (35). It is initially deposited at the posterior pole
behind the primary vitreous, later extending to envelop the entire primary vitreous.
Tertiary vitreous refers to a phase of development around the end of the third month,
where distinct condensations of the secondary vitreous become evident at the lens
equator, associated with the formation of lens zonules, and become firmly attached to
the inner limiting membrane of the retina in the developing pars plana region, forming
the vitreous base. The tertiary vitreous and zonular fibres are actually thought to be
produced by the neuroectoderm of the developing ciliary body (35).
During the 4th month of gestation, regression of the primary vitreous, the hyaloid
vessels, and the tunica vasculosa lentis occurs. The course of the regressing hyaloid
artery is evident in the adult vitreous as Cloquet’s canal, a narrow, fluid-filled central
channel that extends from the optic disc posteriorly to the region of the degenerating
tunica vasculosa lentis anteriorly. Failure of the hyaloid system to regress can result
in developmental abnormalities of the vitreous such as a Mittendorf dot, Bergmeister’s
papilla, persistent hyaloid artery, and persistent fetal vasculature (previously known as
persistent hyperplastic primary vitreous) (35). The adult vitreous is a specialised
extracellular matrix composed of hyaluronan, the major macromolecule, interwoven
with collagen fibrils (composed of collagen types II, IX, and V/XI) (36). As such,
inherited diseases resulting from mutations in genes affecting collagen or hyaluronan
function, such Marfan syndrome (FBN1), Ehlers-Danlos syndrome (COL1A1,
COL1A2, COL5A1 and COL5A2), Stickler syndrome (COL2A1, COL9A1, COL11A1),
29
Knobloch syndrome (COL18A1), Wagner syndrome (VCAN) and HYAL2 syndrome
(HYAL2), also often display ocular features associated with vitreoretinal degeneration,
including vitreous syneresis, vitreous traction, high myopia and retinal detachment (37,
38).
1.3 Genetic studies in communities
The field of community genetics or genomics can be defined as “the art (application)
and science (research) of the responsible and realistic application of health and
disease-related genetics and genomics knowledge and technologies in human
populations and communities to the benefit of individuals therein” (39). Communities
can be defined by their geographical location, ethnic origins, or by their cultural,
religious or socio-economic characteristics. Community genetics aims to bring about
clinical benefits to individuals and families affected by genetic diseases, as well as to
locate individuals within the wider community who may also be at risk of the same
inherited conditions. Communities also benefit through liaison with support
organisations and health authorities to ensure delivery of up-to-date genetic
counselling, screening and clinical care to affected individuals.
Our research team has established collaborations with scientists and healthcare
professionals in several communities globally. There is a strong emphasis on
translating the scientific insights arising from our research studies into improved
healthcare outcomes for communities as well as affected individuals globally through
new therapies and diagnostic strategies as well as refinement of genomic healthcare
policy. This thesis focuses on inherited eye diseases in affected families from Amish,
Pakistan and Palestinian communities.
1.3.1 The Amish and Anabaptist communities
The Amish are a distinct group of traditional rural-living Anabaptist Christians, known
for their simple living, plain dress and separation from modern technologies and
conveniences, whose roots can be traced back to the Protestant Reformation in 16th
century Europe. One group of reformers, the Anabaptists, rejected infant baptism,
believing instead that only adults who had confessed their faith should be baptised (or
30
re-baptised). They also advocated for separation of church and state. Due to these
beliefs, Anabaptist groups were severely persecuted throughout Europe, with many
fleeing to the mountains of Switzerland and southern Germany.
One of the church leaders, a Swiss bishop named Jakob Ammann, sought to revitalise
the Anabaptist movement by suggesting reforms of church practices that enforced
closer links between church discipline and social practices. Amman proposed holding
communion twice a year rather than once, as was the typical Swiss practice, and
suggested that Christians, in obedience to Christ, should wash another’s feet in the
communion service. To promote doctrinal purity and spiritual discipline, Ammann
forbade the trimming of beards and the wearing of fashionable dress, teaching that
church members should dress in a uniform manner. He also believed in a stricter
interpretation of the doctrine of shunning or “Meidung”, a practice based on the New
Testament command not to associate with a church member who was not repentful of
his sinful conduct. Ammann taught that if a member of the church was
excommunicated because of an unrepented sin, they should be completely shunned
or avoided by all church members, including refusal of goods from the offending
individual or refusing to share a meal with them. This practice was not intended to be
a punishment but was instead meant as a lesson to help the individual realise the error
of his ways and to seek forgiveness.
These beliefs however caused a schism between Ammann’s followers and the other
Anabaptists in Switzerland and Alsace, leading to an eventual split from the group in
1693. Ammann’s followers were first called the “Amman-ish” group, later becoming
known as the Amish.
The Amish first began emigrating to North America in the early 18th century to escape
persecution in Europe for their religious beliefs. The Amish had largely been landless
tenant farmers in Europe, so the chance to own their own land was also an attractive
proposition. They first settled near Lancaster County in Pennsylvania (“Penn’s
woods”), attracted by the promise of religious freedom as part of William Penn’s “Holy
Experiment” of religious tolerance, where a large community remains today. The first
wave of Amish migration to the state was between 1736 and 1770 and comprised
around 500 individuals.
31
A second wave of Amish immigrants estimated to be around 3000 in number, occurred
between 1815-1860. Compulsory military service was becoming commonplace in
Europe, and this combined with the economic opportunities in North America, was a
strong driving force to leave Europe and seek a new life in the “New World”. These
Amish immigrants settled in new areas outside Pennsylvania, including Ohio, Indiana,
Iowa and Illinois. The last Amish settlement in Europe disappeared in 1937; none
remain in Europe today.
Today, the Amish are found in 31 states in North America, the Canadian provinces of
Manitoba, New Brunswick, Ontario, and Prince Edward Island, and the South
American countries of Argentina and Bolivia. Nearly two-thirds still live in the three
states where the first Amish settlements were established; Pennsylvania, Ohio and
Indiana (40). All Amish people share common beliefs and practices, including adult
baptism, non-violence, lay ministers, small local congregations with services held in
the homes of members with no formal religious buildings, rural living and separation
from the outside world, education only up to eighth grade, church-regulated and
traditional plain style of dress, selective use of technology, use of horse-drawn
transportation and speaking a German-derived dialect (41). There are however around
40 different Amish affiliations, each comprising groups of church districts united by
social and religious practices, differing with regards to types of dress, rules about
technology and restrictions on participating in American society.
Founder effect and inherited diseases in the Amish
There is unfortunately a higher incidence of specific autosomal recessive genetic
disorders, such as hereditary spastic paraplegia (42) and Ellis-van Creveld syndrome
(a form of dwarfism) (43), among the Amish compared to the general population due
to a phenomenon known as the “founder effect”. This refers to a loss of genetic
variation that occurs when a new population is established by a very small number of
individuals, or founders, from a larger population (Figure 1.5). Rare, recessive
disease-causing mutations, if present in unaffected carrier, may then accumulate and
become enriched in the population as it expands over subsequent generations,
32
leading to an increased frequency of certain otherwise rare autosomal recessive
conditions within these communities (44).
Figure 1.5 Ancestral bottleneck leading to founder effect
The modern Amish community in America is descended from the few hundred
German-Swiss settlers who immigrated to the United States in the 18th century, many
of whom were already part of related family groups. This limited number of ancestors
founded a rapidly expanding Amish population, now estimated to number >350,000 in
North America (40). One of the driving forces behind this population growth is
attributed to large family sizes, with an average of 7-9 children per family. The Amish
tend to have limited geographical mobility due to their religious constraints on
transportation, and they tend to observe strict endogamy, marrying only within the
community, with little or no migration into the community. This geographical and
sociological isolation further contributes to the unique genetic distinctiveness of the
Amish, providing a unique opportunity for the study of rare inherited disorders and
identification of causative disease genes (45).
Genetic studies in the Amish are also aided by cultural and demographic
characteristics of the community. The Amish keep extensive historical and
genealogical records, enabling the construction of comprehensive family pedigrees.
Large nuclear families (with low rates of non-paternity) are common, with a number of
33
unaffected as well as affected siblings with the same condition within each family,
facilitating genetic analyses for identification of the causal disease variant. The impact
of environmental factors on the expression of genetic diseases is minimised by the
high standards of living, homogeneous lifestyles, and uniform socio-economic and
occupational circumstances. Standards of medical care are relatively high, with
extensive availability of medical records. The Amish also show an interest in illness
and in the health of their relatives, with extensive sharing of health issues both locally
and through publications such as “The Budget” newspaper, creating networks of
knowledge about individuals with health problems. They also frequently provide home-
based care for children with birth defects or genetic disorders instead of
institutionalisation (46-48). Whilst studies of rare inherited diseases in modern diverse
populations are hampered by small numbers of affected individuals and genetic and
environmental complexities, these factors enhance the visibility of genetic disorders
within the Amish communities, greatly facilitating the discovery of genes responsible
for these inherited conditions.
Studies of inherited disorders present in the Amish were first undertaken in 1962 by
the world-renowned geneticist Victor McKusick. These studies were inspired by an
article written by a local family doctor, David Krusen, mentioning the frequent
occurrence of achondroplasia among the Amish, as well as a manuscript entitled
“Amish Society” by John Hostetler, which highlighted several characteristics of the
Amish community that were advantageous to the study of genetic traits (47). The early
studies on dwarfism in the Amish led to the recognition that the majority did not in fact
have achondroplasia, and this instead led to the description of two separate forms of
autosomal recessive dwarfism within the community; Ellis‐van Creveld syndrome, a
previously recognised disorder, as well as cartilage-hair hypoplasia, a new form of
metaphyseal chondrodysplasia (47).
Over the next few decades, McKusick and his colleagues published over 30 reports of
genetic disorders among the Amish (49). The work of McKusick led to the development
and publication of the Mendelian inheritance in Man (MIM), first published in print as
a trilogy of catalogues containing overviews of genes and genetic phenotypes for
autosomal dominant, autosomal recessive and X-linked conditions (50). Print editions
have since been superseded by an online version, the Online Mendelian of Inheritance
34
in Man or OMIM, providing a comprehensive and authoritative compendium of human
genes and genetic phenotypes that is updated daily, and is a frequently used and
invaluable resource in the field of medical genetics (51).
The Amish are surprisingly open to participation in genetic studies, and this receptivity
may stem from the initial approaches by physicians and geneticists who were
genuinely interested in helping affected individuals in the community. For instance, in
patients with Ellis‐van Creveld syndrome, arrangements were made for surgical repair
of the associated cardiac defects and for orthopaedic correction of knee deformities.
Researchers working with the community have also reported a willingness for families
to participate in studies, even when their affected children may not necessarily benefit,
because of the potential benefit to others, reflecting their generous and altruistic
nature.
The higher incidence of rare inherited disorders among the Amish places a significant
social and financial burden on this community. Whilst community schemes are
available to help with medical expenses, the rising costs of medical care means that
many families experience difficulties in obtaining clinical investigations and treatments.
The Windows of Hope (WoH) Project was established in 2000, and is a non-profit
translational community genomics research program led by Professor Andrew Crosby
and Dr Emma Baple (research supervisors). It aims to determine the clinical and
molecular spectrum of inherited diseases within the Amish and other Anabaptist
communities by facilitating collaborations between clinical specialities as well as
research and diagnostic genomic laboratories. The project has assisted in the
identification and description of the underlying genetic cause in a large number of
inherited disorders, including 18 novel conditions amongst the Amish and Anabaptist
communities. These findings have allowed numerous families to receive much-needed
diagnoses for previously unrecognised conditions, leading to an improved
understanding of the clinical course and associated features, thereby facilitating
improved clinical and developmental outcomes for the affected individuals through
early medical intervention and social support.
To date, researchers have described over 250 separate genetic disorders among the
Amish and other Anabaptist communities, collated into several community-specific
35
databases (52) (WoH Project Database, viewed at https://wohproject.com/). These
include conditions that are completely new to medical science, and although a few
disorders are unique to the Amish so far, most disease genes initially identified in the
Amish have subsequently been shown to cause similar diseases in other populations
worldwide, highlighting the global importance and relevance of studying inherited
conditions in genetic isolates such as the Amish. There are also rare genetic disorders
described in very small numbers of affected individuals identified in modern diverse
populations, but present at increased frequency in the Amish communities due to an
enrichment of disease-causing mutations. The larger numbers of affected individuals
within the Amish communities facilitates the study of these rare genetic disorders, and
may illuminate otherwise obscure biochemical pathways and enable a more
comprehensive understanding of physiologic processes in human health and disease.
This enhanced knowledge of the genetic and molecular mechanisms underpinning
human disease may in turn lead to the development of new treatment strategies,
thereby benefiting affected individuals worldwide. The increased numbers of affected
individuals with a disease in a community also facilitates clinical studies to determine
the clinical minutia of a condition, and to assess the efficacy of potential treatments as
they are developed.
1.3.2 Inherited diseases in Pakistan
Pakistan is the 5th most populous country in the world, with a population exceeding
216 million (53), and is comprised of four provinces (Punjab, Khyber-Pakhtunkhuwa
or KPK, Sindh and Balochistan) and three territories (Islamabad Capital Territory,
Gilgit-Baltistan and Azad Kashmir). The population of Pakistan is ethnically and
linguistically diverse, with around 18 ethnicities (based on historical lineage,
geographical origin, language and cultural practices) and over 60 spoken dialects (54).
The major ethnic groups are Punjabi and Pashtun (also known as Pathan) in northern
Pakistan and Sindhi, Saraiki, Muhajirs (the immigrants from India, mostly Urdu-
speaking) and Baloch in southern Pakistan (55). Social stratification is strongly based
on ethnic and tribal groupings, with tribal groupings based on the biraderi or
‘brotherhood’ system, a patrilineal kinship that transcends geographical boundaries
and is determined by family background and occupation (56).
36
There is a strong preference for endogamous marriage in Pakistan, where it is
estimated that 40-60% of all marriages occur among relatives, particularly between
first and second cousins (54, 55, 57). This arises in part due to the clan-orientated
nature of the society, which values similarities in social group identity based on
religion, ethnicity and tribal/clan affiliation (54). Cultural preferences for
consanguineous marriages include the belief that the marriage will be more stable and
secure with high fertility and lower divorce rates. There might already be existing family
relationships between partners who are likely to have similar socioeconomic status
and family customs, and there are financial benefits such as strengthening of the
family business and maintenance of property within the family (55, 58). Most marriages
still occur within the biraderi, with inter-biraderi marriages rare due to social
stratification and limited social mixing (56). The frequency of autosomal recessive
disorders is higher in populations with a high degree of endogamy, with Pakistan
having one of the highest rates of genetic disorders in the world (59). The high rates
of traditional intra-familial marriages, strong socio-cultural and ethnic divides, and
geographical barriers and isolation has led to numerous genetically distinct
communities, where there may be a high prevalence of specific inherited disorders
due to the accumulation of regional founder variants (60, 61). These founder mutations
often represent important causes of disease in a particular region, and knowledge of
their presence, frequency and clinical outcomes will lead to enhanced knowledge of
the genomic architecture of inherited diseases in Pakistan. This knowledge is of
enormous value for local healthcare resource planning and will facilitate the design of
community-specific hierarchical strategies for cost-effective genetic testing arrays to
enable an accurate disease diagnosis to be achieved more rapidly, thus aiding the
development of diagnostic and clinical care pathways and policies throughout Pakistan
(59). This has led to the early development of specific genomic databases correlating
disease mutation with clinical phenotype, geographic localisation and ethnicity or tribal
affiliation within Pakistan (57).
1.3.3 Inherited diseases in Palestine
The Palestinians are a group of people who either live in, or originate from, historical
Palestine, which formed the land bridge between the continents of Europe, Asia and
Africa. Control of the region is strategic, and it remains one of the most contested
37
regions of the world today. Creation of the state of Israel in 1948 resulted in a
displacement of a large proportion of the Palestinian Arab population, mainly to the
West Bank and Gaza, but also to neighbouring Arab countries including Jordan,
Lebanon and Syria. The Oslo accord of 1993 subsequently resulted in the
establishment of Palestinian autonomy in the Occupied Palestinian Territories of the
West Bank and Gaza. The global Palestinian population is now estimated to be 13.4
million, with the Palestinian diaspora numbering 6.7 million; 5.0 million in the Occupied
Palestinian Territories, and 1.6 million in Israel (62). The majority of Palestinian Arabs
are Muslims (over 80%); other religious communities are smaller and include
Christians and the Duze (63).
Most of the Palestinian Arabs in Israel and in the territories under the Palestinian
Authority live in villages or tribes that were founded by only a few individuals. With
many Palestinians having large families (in 1993, the average number of offspring per
woman in the West Bank and Gaza was 7.8) (64), there has been a natural expansion
of the Arab population. Consanguineous marriages are common among the
Palestinian Arab population, with over 40% of marriages occurring between relatives,
and of these approximately half are between first cousins, most often involving children
of brothers (65). Middle Eastern societies, and in particular the rural Arab populations,
are characterised by close family relationships, and the preference for marrying
relatives is a deeply rooted cultural trait. Although there appears to be evidence for a
declining trend in consanguineous marriages amongst Palestinian Arabs (66-68), they
remain prevalent within this population due to a number of political, economic and
social factors that favour such marriages. Each village or tribe can therefore be
considered a genetic isolate, where autosomal recessive diseases are relatively
frequent, as the introduction of a new recessive mutation can lead to a relatively rapid
appearance of several affected individuals in only a few generations. Whilst some
inherited disorders such as thalassaemia and familial Mediterranean fever are
prevalent throughout the Palestinian Arab population (69), the distribution of
autosomal recessive diseases in general is not uniform, with many rare inherited
diseases, including some novel diseases, specific to a single tribe or village due to this
local founder effect (63). Inherited diseases are an important cause of childhood
morbidity and mortality in these communities, however, particularly in the rural
Palestinian areas, families often do not have access to advanced medical diagnostics
38
or assessment by trained specialists, and an accurate clinical diagnosis is often
difficult to establish. A detailed knowledge of the distribution of genetic diseases in
each village can therefore enable appropriate medical management as well as genetic
counselling to be provided within the community (69).
1.4 Inherited eye diseases
Inherited eye diseases refer to a clinically and genetically heterogeneous group of
disorders that can affect the structure and/or function of one or several structures of
the eye, such as the cornea, anterior segment, lens or retina.
A large number of inherited eye disorders have been identified; these include
conditions limited to the eye, as well as complex disorders such as chromosomal
abnormalities or genetic syndromes with ocular manifestations that are associated
with other systemic anomalies such as hearing impairment, learning disability or
neurological deficits. The eye is an organ that is easily accessible for examination, and
can provide a diagnostic window for wider systemic diseases; examples include the
cherry red spot seen in Tay-Sachs and Sandhoff disease, angioid streaks in
pseudoxanthoma elasticum, the iridescent “Christmas tree” cataract in myotonic
dystrophy, lens subluxation in Marfan syndrome and homocystinuria, or the whorl-like
corneal opacities or corneal verticillata seen in Fabry disease. Whilst inherited eye
diseases are rare individually, together they contribute significantly to the burden of
visual impairment and blindness.
Visual impairment is a major public health problem worldwide, with the World Health
Organisation (WHO) estimating that in 2010, there were 285 million people globally of
all ages who were visually impaired, of whom 39 million were blind (70) [according to
the International Classification of Diseases Update and Revision 2006, visual
impairment is classified as a visual acuity of less than 6/18, whilst a visual acuity of
less than 3/60 would be classified as blindness (Snellen values) (71)]. In response to
this global need, the WHO, in collaboration with the International Agency for the
Prevention of Blindness (IAPB), launched the VISION 2020: The Right to Sight global
initiative, aiming to eliminate avoidable blindness in the world by the year 2020, with
the control of blindness in children (under age 16) a high priority (72, 73).
39
Inherited eye diseases contribute significantly to the burden of childhood blindness,
accounting for approximately 40-50% of all childhood blindness in industrialised
countries and 20-30% in developing nations (although this likely represents an
underestimate due to poor clinical facilities and access in some regions) (73). Globally,
the main inherited eye diseases that cause visual impairment are retinal dystrophies,
corneal dystrophies, congenital and juvenile cataracts, aniridia and albinism (74). In
the UK, inherited eye diseases account for a third of all children with severe visual
impairment or blindness, with retinal dystrophies, microphthalmia/anophthalmia,
nystagmus and albinism being important hereditary causes (75, 76). Hereditary retinal
disorders are the most common cause of certifiable visual impairment in working age
adults (accounting for a fifth of certifications in this age group) (77), and the third most
common cause of certifiable visual impairment overall in the UK (78).
Blindness is a major disability that impacts on the physical, mental and social health
of affected individuals, and places a significant emotional and financial burden on
affected families and the wider community. As individuals with isolated inherited eye
diseases often develop visual impairment at a young age and have a normal life
expectancy, these conditions are associated with significant morbidity and socio-
economic impact. Childhood blindness is a particularly significant problem in
developing countries because many families to not have adequate access to
healthcare and ophthalmic services due to a lack of infrastructure, resources and
funding in some regions.
Establishing a precise clinical and molecular diagnosis in individuals with inherited eye
diseases usually involves a thorough clinical examination to delineate the ocular
phenotype and establish the presence or absence of any associated systemic
features, a genetic workup including pedigree construction to show medical histories
and genetic relationships within a family that might suggest a possible mode of
inheritance, followed by chromosomal or molecular analysis to try identify the
underlying genetic aetiology. An accurate disease diagnosis can bring about many
important benefits for both patients and their families. It improves understanding of the
disease which aids clinical management decisions and provides useful prognostic
information for affected families. It can highlight the need for periodic screening to
40
enable early diagnosis or facilitate early treatment to prevent visual morbidity, such as
prophylactic laser or cryotherapy treatment to prevent retinal detachment in individuals
with Stickler syndrome (79). An accurate molecular diagnosis enables informed
genetic counselling about the medical implications for the affected individual and
members of the wider family, and it may provide advice on recurrence risk crucial for
couples who wish to have more children. Determination of carrier status may provide
reassurance that the risk of having affected children is low, or it may highlight a need
for prenatal diagnostic evaluation and follow-up.
Determining the underlying genetic diagnosis in inherited eye diseases however is
often challenging due to the phenotypic and genetic heterogeneity of these conditions.
For instance, pleiotropic disorders such as Bardet-Biedl syndrome (BBS) can create
diagnostic difficulties due to the multiple, seemingly unrelated phenotypic
manifestations of the disease (80). Some inherited eye diseases such as inherited
retinal dystrophies demonstrate remarkable genetic heterogeneity with overlapping
clinical phenotypes; to date there are over 271 disease-associated genes (RetNet
https://sph.uth.edu/retnet/, accessed 28.04.2021), spanning the entire spectrum of
Mendelian (autosomal dominant, autosomal recessive and X-linked) and non-
Mendelian (mitochondrial and digenic) inheritance (Figure 1.6). Recent advances in
molecular diagnosis have also highlighted numerous inaccuracies in the traditional
phenotype-based corneal dystrophy classification due to the genetic and phenotypic
heterogeneity associated with the condition. For instance, mutations in two separate
genes (KRT3 and KRT12) have both been shown to cause the Meesman dystrophy
phenotype, whilst several distinct corneal dystrophy phenotypes including Reis-
Buckler, Thiel-Behnke, granular, lattice and Avellino corneal dystrophies have now all
been shown to be caused by mutations in the same TGFBI gene. This has led to a
revised classification by the International Committee for Classification of Corneal
Dystrophies (IC3D) incorporating this new genetic understanding of corneal
dystrophies, with TGFBI-associated dystrophies now classified into a single grouping
(81, 82).
41
Figure 1.6 Genes associated with inherited retinal dystrophies
The coloured circles represent clinical diagnoses; overlapping circles indicate the significant clinical and genetic heterogeneity associated with inherited retinal dystrophies. Abbreviations: COD, cone dystrophy; CORD, cone-rod dystrophy, CSNB, congenital stationary night blindness, LCA, Leber congenital amaurosis; MD, macular dystrophy, RP, retinitis pigmentosa; VR, vitreoretinopathy. Modified from Berger et al (83)
Establishing an accurate clinical and molecular diagnosis is a particularly significant
problem in developing countries, where there is limited access to ophthalmic
equipment and expertise needed for performing and interpreting the detailed
phenotyping studies needed for diagnosis due to regional geographical limitations or
restricted local clinical resources. This problem is further compounded by a relative
lack of knowledge regarding the specific nature and causes of inherited ocular
diseases in developing nations.
Advances in next generation sequencing (NGS) technologies have ushered in a new
era in molecular diagnostics by facilitating the sequencing of whole exomes or
genomes of large cohorts of individuals with inherited eye diseases. This has not only
42
led to the identification of numerous new genetic causes responsible for a wide
spectrum of inherited eye diseases such as corneal dystrophies, retinal dystrophies,
and ocular developmental defects (84, 85), but may also permit a hypothesis-free
diagnostic approach in settings where clinical information is limited, a situation
commonly encountered in developing nations due to geographical isolation and limited
access to healthcare services (see thesis chapter 5.3). There is a global responsibility
to support genomic health initiatives in these nations so that the transformative
potential of genomic medicine can be translated into meaningful health outcomes that
benefit populations worldwide (59, 86).
1.4.1 Gene therapy in inherited eye diseases
Inherited eye diseases have historically played an important role in defining the basic
principles of genetics. In 1876, the Swiss ophthalmologist Johann Friedrich Horner
described the characteristic X-linked recessive pedigree pattern in a family with colour
blindness, furthering progress in the understanding of sex-linked heredity; Horner’s
law stated that “colour-blind fathers have colour-normal daughters; and these colour-
normal daughters are the mothers of colour-blind sons” (87). In 1936, Imai and
Moriwaki first suggested cytoplasmic inheritance (now known as mitochondrial
inheritance) to account for the maternal inheritance pattern of Leber hereditary optic
neuropathy (88). The patchy retinal pigmentary pattern seen in heterozygote carriers
of X-linked ocular albinism was seen to support the Lyon hypothesis, which postulated
the random inactivation of one of the X chromosomes in somatic cells of females (89).
Knudson’s observations on retinoblastoma led to the development of the two-hit
hypothesis in 1971, fundamental to our understanding of tumour suppressor genes
and familial cancer syndromes (90). And the first convincing report of digenic
inheritance in human disease was in 1994 for retinitis pigmentosa caused by mutations
in the PRPH2 and ROM1 genes (91).
Today, advances in molecular genetics have in turn contributed significantly to
ophthalmic genetics, advancing our understanding of the molecular biology of eye
development and vision and the pathophysiological basis of eye diseases. For
instance, mechanistic dissection of photoreceptor degeneration in retinal dystrophies
identifies genes affecting a wide variety of cellular functions, including ciliary transport,
43
phototransduction and the visual cycle, lipid oxidation, protein degradation, cell
signalling and cell-cell interactions, intracellular transport, phagocytosis and RNA
splicing (92). Knowledge of the underlying genetic and molecular causes of disease
can allow redeployment of existing therapies in targeting the biochemical pathways
involved in disease pathogenesis, which may slow or halt disease progression, or
enable restoration of protein or cell function through gene therapy or other
experimental approaches.
The eye has a number of advantages as a target organ for gene therapy (93, 94). It is
easily accessible by surgical approaches, and ocular tissues can be imaged and
quantified in vivo with non-invasive imaging techniques such as electroretinography.
Due to its small anatomical size and subdivision into smaller compartments, only small
volumes of gene delivery vectors need to be administered to ensure delivery to target
tissues. The relatively tight blood-retina barrier allows concentration of vectors within
the target area and limits potential complications from systemic exposure, whilst at the
same time altering the trafficking of immune cells from the systemic circulation to the
eye and providing an “immune-privileged” state that limits the immune reaction to a
given gene vector. The duplicity of the eye as an organ, and the bilateral nature of
many inherited eye diseases, allows the option of treating just one eye, and
considering the untreated eye as an ideal within-subject experimental control for
comparisons on the safety and efficacy of treatment.
Gene therapy approaches have mainly focused on adeno-associated virus (AAV) or
lentivirus vector-based gene replacement strategies in inherited retinal dystrophies,
conditions for which management options were previously limited to visual
rehabilitation, educational and social support. The recent approval of Luxturna®
(voretigene neparvovec) for the treatment of RPE65-mediated inherited retinal
dystrophy in Europe and the United States (95, 96), together with early promising
results from clinical trials currently underway for a further range of retinal dystrophies
associated with CHM, PDE6B, RPGR, MERTK, MYO7A, ABCA4, RS1, CNGA3 and
CNGB3 mutations (94), marks a new era in the treatment of these previously incurable
and sight depriving conditions. Non-viral systems for gene delivery such as
electroporation, nanoparticles and liposomes are also being studied, and although
44
these will be cheaper and easier to produce, they currently do not seem to show the
same therapeutic promise (94).
Antisense oligonucleotides are another promising therapeutic strategy that can be
used to target disease mutations, with several different approaches currently
undergoing clinical trials in inherited retinal dystrophies. One approach uses the
antisense molecule to block the activation of cryptic splice sites and restore normal
splicing mechanisms, and is useful for diseases resulting from aberrant splicing; this
approach is currently under investigation for the treatment of Leber congenital
amaurosis due to a splice site mutation in the CEP290 gene (97). Another approach
based on splice modulation is being studied for the treatment of syndromic and non-
syndromic retinitis pigmentosa caused by mutations in the USH2A gene; a common
site of disease mutations is within exon 13 of this gene, and the antisense molecule is
designed to cause skipping of this exon from the USH2A mRNA, resulting in an in-
frame transcript that is translated into a shorter but still functional usherin protein (98).
A further approach can be used for diseases caused by dominant negative mutations,
where the antisense molecule targets and degrades the mutant mRNA; this approach
is currently being trialled for the treatment of autosomal dominant retinitis pigmentosa
caused by RHO gene mutation (99).
Another gene-based therapeutic strategy under investigation is the use of small
molecule translational read-through inducing drugs or TRIDs such as PTC124
(Ataluren) or aminoglycoside antibiotics such as gentamicin (G418). These act by
interfering with the proof-reading abilities of translationally active ribosomes, allowing
“read-through” of premature termination codons (PTCs) and translation of a full-length
protein from the mutant mRNA. This approach may be useful for recessive diseases
where nonsense mutations are a significant cause of disease, and where a small
amount of functional protein may be sufficient to rescue the disease phenotype, and
has been studied for a number of inherited ocular conditions including choroideraemia,
retinitis pigmentosa, Usher syndrome and aniridia (100). TRIDs have several
advantages compared to gene replacement strategies; the size of the gene is not
crucial (compared to the limited packaging capacity of commonly used viral vectors),
and the targeted genes remain under endogenous control, maintaining tissue-specific
45
timing and duration of gene expression and splicing. Concerns remain however over
long-term toxicity and the need for repeated dosing.
Recently, the first in-human trial of genome editing using the clustered regularly
interspersed palindromic repeat (CRISPR)/Cas9 system has been performed to target
and delete a cryptic splice site and restore normal splicing in individuals with Leber
congenital amaurosis (101), with plans for a similar trial targeted to Usher syndrome
(102, 103).
Although progress is not as advanced as for inherited retinal dystrophies, gene therapy
approaches are also being investigated as a therapeutic option for other inherited eye
diseases including Leber hereditary optic neuropathy (104), mucopolysaccharidoses
(105), Fuchs corneal endothelial dystrophy associated with expansion of a non-coding
trinucleotide repeat in TCF4 (106), and MYOC-associated glaucoma (107).
Challenges remain in the development of molecular therapies for inherited eye
diseases, both in terms of technical hurdles, and the social responsibility to provide
equitable access to these transformative therapies given the significant human cost of
sight loss for affected individuals (108). Despite this, advances in molecular
technologies have greatly expanded the diagnostic capabilities and therapeutic
landscape for inherited eye diseases, providing an optimistic outlook for individuals
with these previously untreatable conditions.
1.5 Disease gene and variant identification strategies
Identification of disease genes enables an accurate molecular diagnosis in affected
individuals, resulting in improved clinical care. It is also crucial to understanding the
underlying genetic and molecular pathways of disease, which may in turn highlight
potential therapeutic targets. The rapid evolution and widespread introduction of next
generation sequencing (NGS) technologies such as whole exome and whole genome
sequencing in research as well as clinical diagnostic settings has allowed rapid
progress in the identification of genes and variants associated with inherited eye
diseases. The combined approach of NGS in parallel with autozygosity mapping, a
powerful tool in the study of autosomal recessive diseases in endogamous
46
communities, has been successful in uncovering novel causes of inherited eye
diseases in community settings (38, 60), and was the primary approach adopted in
many studies outlined in this thesis. Further background information on autozygosity
mapping and advances in NGS technologies is contained in Appendix A
1.6 Project aims
The overarching objective of this PhD project involves the delineation and
characterisation of the phenotypic, genetic and molecular spectrum of inherited eye
diseases in community settings including the Amish, Pakistani and Palestinian
communities. This will increase scientific understanding of the genetic causes and
molecular pathways underlying ocular diseases, and drive advances that will benefit
patients worldwide with the same condition. In order to achieve this, the specific aims
of the study include:
1. screening of established disease genes in affected families with isolated and
syndromic inherited eye diseases including: anophthalmia, infantile nystagmus
and oculocutaneous albinism (OCA), anterior segment dysgenesis and
congenital cataracts, as well as inherited retinal dystrophies to identify
previously reported and novel pathogenic gene variants. Alongside this,
comprehensive literature reviews will be undertaken to determine the clinical
and molecular spectrum of inherited eye diseases in communities, facilitating
the development of targeted genetic testing strategies that will enable improved
and efficient disease diagnosis and early intervention,
2. investigation of the pathogenicity of two common TYR gene variants through
co-segregation studies in Amish OCA families, together with analysis of
extensive UK and international OCA patient cohorts and functional assays. This
will provide novel insights into OCA molecular pathogenesis, and help resolve
a long dispute regarding the pathogenicity and clinical relevance of these gene
variants,
47
3. perform detailed clinical phenotyping in combination with comprehensive
genomic studies in molecularly undiagnosed individuals with inherited eye
diseases. This will enable the identification, consolidation and characterisation
of ultra-rare causes of inherited eye diseases in Amish, Pakistani and
Palestinian communities.
48
2 MATERIALS AND METHODS
2.1 Materials
General laboratory consumables were purchased from Rainin, STARLAB and Alpha
Laboratories. Reagents used are reported in Table 2.1.
Table 2.1 Reagents used in the study
Reagents Manufacturer
Agarose (molecular grade) Fisher Scientific
Boric acid Fisher Scientific
dNTP set Solis BioDyne
DMSO Sigma-Aldrich
DreamTaqTM DNA polymerase Thermo Fisher Scientific
DreamTaqTM green buffer (10x) Thermo Fisher Scientific
Ethanol Fisher Scientific
Ethidium bromide Fisher Scientific
Exonuclease I (Exo I) New England Biolabs
GeneRulerTM 1kb Plus DNA ladder Thermo Fisher Scientific
Lithium acetate dihydrate Alfa Aesar
Shrimp alkaline shosphatase (rSAP) New England Biolabs
2.2 Clinical methods
2.2.1 Ethical approval for study
Ethical approval was granted from the ethical approval committees of:
• University of Exeter Medical School, Exeter, UK
• South Central – Hampshire A Research Ethics Committee, Southampton, UK
• Akron Children’s Hospital, Ohio, USA
• Baylor College of Medicine, Texas, USA
• University of Arizona, Arizona, USA
• International Islamic University, Islamabad, Pakistan
• Kohat University of Science and Technology, KPK, Pakistan
• University of Health Sciences, Lahore, Pakistan
49
• Shah Abdul Latif University, Sindh, Pakistan
• Arab American University, Jenin, Palestine
The study protocol adhered to the tenets of the Declaration of Helsinski. Written
informed consent was obtained from all participants prior to their inclusion in this study
with parental written consent provided on behalf of children involved in the study.
2.2.2 Patient ascertainment and phenotyping
Recruitment of families from the North American Amish communities, as well as
communities in Pakistan and Palestine, was performed by collaborating researchers.
All study participants were phenotypically assessed and overseen by local clinicians.
A medical history was obtained, including a documentation of visual symptoms. Facial
photographs and videos were used to document skin and hair tone and nystagmus.
Visual acuity testing was performed using Snellen charts, and colour vision testing
using Ishihara charts. The anterior and posterior segment of the eye was examined by
slit lamp biomicroscopy or direct fundoscopy. Additional investigations were performed
in selected affected individuals where possible; these include retinal imaging studies
such as colour fundus photography and ocular coherence tomography (OCT), as well
as electrophysiological testing including electroretinography. Subsequent to molecular
genetic studies, additional targeted clinical investigations were occasionally
undertaken to clarify the clinical significance of any candidate variants identified.
Considerable diagnostic difficulties were encountered by clinicians in some
communities, where affected families resided in rural regions with a lack of medical
infrastructure, limiting access to specialised equipment required for detailed and
accurate ocular phenotyping. Local clinicians and scientists were supported by myself
(clinical ophthalmologist) in the collection and review of the highest quality clinical
phenotypic data possible.
50
2.3 Molecular genetic methods
2.3.1 Sample acquisition and data management
The research study was carried out in compliance with the Human Tissue Authority
(HTA) Codes of Practice and Standards (Code E: Research). All blood and buccal
samples and subsequent DNA extractions used in this project were stored in HTA-
licensed premises with research carried out in accordance with the Human Tissue Act
2004. Each participating individual was assigned a unique study identification number.
A master list of study participants linking the identification number to the study
individual was recorded in a password protected database and stored on university
servers. All data management was GDPR compliant.
Participating individuals had either peripheral venous blood samples taken in EDTA-
containing vacutainer tubes, or buccal cell collection using the ORAcollect® for
paediatrics kit (DNA Genotek). Blood and buccal sample tubes were labelled with their
study identification number on arrival; blood samples were stored at -20°C, whilst
buccal samples were stored at room temperature, prior to DNA extraction.
2.3.2 DNA extraction and quantification
DNA extraction from whole blood
Genomic DNA extraction for venous blood samples was performed using the
ReliaPrepTM Blood gDNA Miniprep System (Promega). Blood samples were
thoroughly mixed by hand for at least 2 mins. 200 μl of blood was added to 20 μl of
proteinase K solution in a 1.5 ml microcentrifuge tube and mixed briefly. 200 μl of cell
lysis buffer was then added to the tube, which was capped and mixed by vortexing
(Topmix FB15024 Vortex Mixer, Fisher Scientific) for at least 10 sec, followed by an
incubation for 10 mins at 56°C on a heating block (Mixer HC Thermoblock, STARLAB).
After this incubation period, the tube was removed from the heating block, and 250 μl
of binding buffer was added to the tube, which was then capped and mixed for 10 sec
using a vortex mixer. At this stage, the lysate was visually checked to ensure a dark
green colour as per protocol specifications.
51
The contents of the tube were then added to a ReliaPrepTM binding column placed in
an empty collection tube, which was capped and centrifuged for 1 min in a
microcentrifuge (Biofuge Pico Microcentrifuge, Heraeus) at maximum speed (13,000
rpm, 16,060 g-force). The binding column was checked to make sure that the lysate
had completely passed through the membrane, and if there was lysate still visible on
top of the membrane, the column was centrifuged for a further 1 min. The collection
tube containing the flow-through was then removed, and the liquid discarded as
hazardous waste. The binding column was placed in a fresh collection tube, and 500
μl of column wash solution was added to the column, this was then centrifuged for 3
mins at maximum speed, and the flow-through discarded. This last step was repeated
twice more for a total of three washes.
The column was then placed in a clean 1.5ml microcentrifuge tube, and 200 μl of
nuclease free water was added to the column. This was centrifuged for 1 min at
maximum speed to elute the DNA.
DNA extraction from buccal cells
Genomic DNA extraction for buccal samples was performed using the Xtreme DNA kit
(Isohelix). The sample tubes containing the stabilised swab heads were first incubated
in a water bath at 50°C for 1 hour. 20 μl of proteinase K solution was then added to
the sample tube, which was immediately mixed by vortexing (Topmix FB15024 Vortex
Mixer, Fisher Scientific), and then further incubated in a water bath (Unstirred Digital
Water Bath, Clifton) at 60°C for 1 hour. 750 μl of column binding buffer was added to
the tube, which was mixed by vortexing for 30 sec. 1.25ml of ethanol was then added
to the tube, and this was mixed by vortexing.
700 μl of the sample was added to an Xtreme DNA column placed in a collection tube,
and this was centrifuged for 1 min in a microcentrifuge (Biofuge Pico Microcentrifuge,
Heraeus) at maximum speed (13,000 rpm, 16,060 g), with the flow-through then
discarded. This step was repeated until all the sample had been loaded onto the
column. 750 μl of wash buffer solution was then added to the column, and this was
centrifuged at maximum speed, with the flow-through discarded. This step was
52
repeated once for a total of 2 washes. The column was then placed in a clean
collection tube and centrifuged at maximum speed for 3 mins to remove all traces of
ethanol.
Following this, the column was placed in a clean 1.5 ml microcentrifuge tube and 100
μl of elution buffer (preheated to 70°C) was added to the centre of the membrane. The
column was allowed to stand for 3 mins, following this it was then centrifuged at
maximum speed for 1 min to elute the DNA.
DNA quantification and storage
Extracted DNA was quantified using a spectrophotometer (NanoDropTM 2000, Thermo
Fisher Scientific) to measure the absorption of UV light at 260 nm (peak absorbance
wavelength of nucleic acids) in a 1 μl sample aliquot. The NanoDropTM software
automatically calculates the nucleic acid concentration (in ng/μl) using a modified
Beer-Lambert equation, which correlates the calculated absorbance with
concentration.
The quality of the extracted DNA was also assessed simultaneously by measuring the
absorption of UV light at 280 nm (strong absorbance wavelength of proteins and
phenolic compounds) and 230 nm (strong absorbance wavelength of organic
compounds). The absorbance at 260 nm, 280 nm and 230 nm normalised to a 10 mm
pathlength is denoted by A260, A280 and A230 respectively, and the A260/A280 and
A260/A230 ratios were used to assess DNA purity. A A260/A280 ratio of ~1.8
generally indicates “pure” DNA; an appreciably lower ratio indicates the possible
presence of protein or other contaminants that absorb strongly at or near 280 nm. The
A260/A230 ratio is a secondary measure of nucleic acid purity, with values for “pure”
nucleic acid often higher than the respective A260/A280 values and commonly in the
range of 1.8 - 2.2; an appreciably lower ratio may indicate the presence of co-purified
contaminants. Following extraction and quantification, DNA was stored at 4°C.
53
2.3.3 Polymerase chain reaction (PCR) and dideoxy sequencing
Primer design
Oligonucleotide primer pairs for dideoxy sequencing were designed using reference
genomic sequences accessed from the UCSC Genome Browser
(https://genome.ucsc.edu/) and the open-source software Primer3Plus
(http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi) for primer design.
Occasionally, it was necessary to design some primers by eye where the regions
flanking the target sequence were highly repetitive or GC rich and hence not conducive
to automatic primer design. Primers were selected based on the following principles:
• Primer size between 20-26 nucleotides in length
• Similar predicted melting temperatures for the primer pair
• Primers that specifically anneal only to the region of interest with no alternative
binding sites in the genome identified [checked using the human BLAT search
tool at UCSC Genome Browser (https://genome.ucsc.edu/cgi-bin/hgPcr)]
• Primers that did not contain any common single nucleotide polymorphisms
(SNPs) (≥1% minor allele frequency or MAF in dbSNP build 151) or repetitive
elements (including low-complexity sequences and interspersed repeats)
identified using the RepeatMasker software tool [checked using the human
BLAT search tool at UCSC Genome Browser (https://genome.ucsc.edu/cgi-
bin/hgPcr)]
• A PCR product size of between 200 to 1000 bp in length (where possible)
• An PCR product GC content of less than 60% (where possible)
Oligonucleotide primers were ordered and manufactured by Integrated DNA
Technologies IDT™ and were received in lyophilised form. Primers were re-
suspended in double-distilled water (ddH2O) as per manufacturer’s instructions to
achieve a 100 μM stock solution and stored at -20°C. A subsequent 5 μM working
dilution of 200 μl was made for PCR amplification and sequencing, and was stored at
4°C. Specific primer details are contained within Appendix D.
54
PCR conditions
PCR reactions for each primer pair were first optimised using a temperature gradient
protocol to determine the optimal primer annealing temperature for achieving a high
yield of PCR product with minimal non-specific amplification (Figure 2.1). The
temperature gradient would typically be run across 12 reactions, with each reaction
having a different annealing temperature ranging from 52-64°C, each differing by
approximately 1°C.
Figure 2.1 Temperature gradient optimisation example
Agarose gel electrophoresis image of PCR reaction using primers designed to amplify the OCA2 p.(Ala787Val) variant. 58°C was the optimal annealing temperature chosen as it was the lowest temperature with a bright and clear band.
PCR was performed using a 10 μl reaction mixture (Table 2.2). The DreamTaqTM
Green Buffer contains MgCl2 (at a concentration of 20 mM) and is optimised for PCR
reaction with the DreamTaqTM DNA Polymerase, the standard Taq polymerase used
in the lab. The buffer also contains a density reagent and two tracking dyes, allowing
direct loading and visualisation of PCR products onto the agarose gel, as well as
facilitating the monitoring of the electrophoresis progress. The dNTP Mix solution was
prepared from stock solutions of 100mM dATP, dCTP, dGTP and dTTP (dNTP Set,
Solis BioDyne).
55
Table 2.2 Standard PCR reaction mixture
Component (concentration) Volume
Primer forward (5 μM) 0.4 μl
Primer reverse (5 μM) 0.4 μl
DreamTaqTM green buffer (10x) 1.0 μl
DreamTaqTM DNA polymerase (5 units/μl) 0.1 μl
dNTP mix solution (10 mM) 0.4 μl
DNA (10-30 ng/μl) 0.8 μl
ddH2O 6.9 μl
Total 10 μl
Modifications to the standard PCR reaction mixture were occasionally required. For
instance, where the PCR products had an unavoidably high GC content (typically
>60%) and where there was non-specific priming or poor PCR product yield, the PCR
reaction was supplemented with the addition of 10% DMSO. In DNA templates with a
high GC content, there is increased difficulty in denaturing the template due to the
increased hydrogen bond strength, causing intermolecular secondary structures to
form more readily, which can compete with primer annealing (109). DMSO is thought
to resolve secondary structure formation by binding to the major and minor grooves of
the template DNA, hence destabilising the double helix structure and promoting
denaturation (109).
PCR was performed in a thermal cycler (Mastercycler® ep gradient S, Eppendorf)
following a touchdown PCR protocol (Table 2.3). This method aims to increase
specificity of the PCR reaction by reducing off-target priming, and uses an initial
annealing temperature (Ta) that is greater than the projected melting temperature of
the primers, followed by a gradual decrease in annealing temperature over
subsequent cycles until the desired annealing temperature (as identified through
temperature gradient optimisation) is achieved (110).
56
Table 2.3 Touchdown PCR protocol
Step Temperature Time
1 Initial denaturation 95°C 5 mins
2 Denature 95°C 30 sec
3 Anneal Ta + 4°C 30 sec
4 Extension 72°C 30-60s
Repeat steps 2-4 for a total of 2 cycles
5 Denature 95°C 30 sec
6 Anneal Ta + 2°C 30 sec
7 Extension 72°C 30-60 sec
Repeat steps 5-7 for a total of 2 cycles
8 Denature 95°C 30 sec
9 Anneal Ta 30 sec
10 Extension 72°C 30-60 sec
Repeat steps 8-10 for a total of 35 cycles
11 Final extension 72°C 5 mins
Abbreviation: Ta, annealing temperature.
The duration of the extension step was modified depending on the size of the PCR
product; for product sizes of less than 500 bp in length, an extension time of 30 sec
was used, for PCR product sizes between 500 to 750 bp long, an extension time of 45
sec was used, and where the PCR product was over 750 bp in length, an extension
time of 60 sec was used. Specific PCR conditions for each primer pair are contained
within Appendix D.
Agarose gel electrophoresis
In agarose gel electrophoresis, the agarose gel forms a highly cross-linked matrix
through which the negatively charged DNA molecule is forced to migrate in response
to an electric current towards the positive anode. This technique can be used to
separate DNA fragments by size, as shorter DNA molecules will migrate more rapidly
through the matrix compared to longer molecules, and will travel a greater distance
across the agarose gel in a given period of time. In this study, agarose gel
electrophoresis was mainly used to determine the adequacy of the DNA amplification
following PCR and to confirm the presence of a single amplicon.
57
1% agarose gels were made by heating 1.0 g molecular grade agarose powder with
100 ml 1X lithium acetate borate (LAB) buffer (consisting of 10 mM lithium acetate and
10 mM boric acid, made by dissolving 51 g of lithium acetate dehydrate and 31 g boric
acid in 1 litre of distilled water to make a 1 litre 50X stock solution, before being diluted
to 1X) in a 700 W microwave till the agarose has completely dissolved. The agarose
solution was left to cool to approximately 50-60°C before 5 μl of 1% ethidium bromide
(a DNA-binding fluorophore) was added, this was then poured into a gel tray with a
well comb in place, and left to sit at room temperature for approximately 30 to 60 mins
until the gel was completely solidified.
The agarose gel was then placed in an electrophoresis tank containing 1X LAB buffer,
and 3 μl of PCR product was loaded directly into the wells (no loading buffer was
required due to the use of DreamTaqTM Green Buffer for the PCR reaction), together
with an appropriate molecular weight marker, most typically the GeneRulerTM 1kb Plus
DNA Ladder, to allow sizing of the PCR product.
The gel was run at 150 V constant voltage for approximately 30 mins, and then imaged
using an ultraviolet transilluminator, causing fluorescence of the ethidium bromide and
allowing visualisation of the DNA banding pattern. The image was photographed using
a gel imaging and analysis system (InGenius gel documentation system and
GeneSnap image acquisition software, Syngene).
PCR clean-up and dideoxy sequencing
Prior to sequencing, unincorporated primers and nucleotides were removed using a
combination of the hydrolytic enzymes exonuclease I (Exo I) (which degrades single
stranded DNA including residual primers and superfluous PCR products) and shrimp
alkaline phosphatase (rSAP) (which dephosphorylates and hence deactivates the
remaining nucleotides) (111).
An ExoSAP enzyme mix was first prepared, consisting of 2.5 μl of Exo I (20,000 U/ml)
and 25 μl rSAP (1000 U/ml), made up to 1 ml with ddH2O. 2 μl of this ExoSAP enzyme
mix was then added to 5 μl of PCR product, and this mixture was incubated at 37°C
58
for 30 mins (for digestion of excess primers and dephosphorylation of nucleotides),
followed by a second incubation at 95°C for 5 mins for enzyme inactivation.
The ExoSAP treated PCR products were sequenced by Source BioScience
(https://www.sourcebioscience.com/), with the generated chromatogram data file
visualised using a chromatogram viewer (FinchTV, Geospiza).
2.3.4 Single nucleotide polymorphism (SNP) genotyping
Within community settings, affected individuals for autosomal recessive diseases are
typically homozygous for founder mutations that occur at an increased frequency
within such populations, and reside within identical-by-descent haplotypes (60, 112).
Homozygosity mapping techniques, which highlight shared regions of homozygosity
between affected individuals, therefore facilitate an efficient means of identifying
candidate disease genes within these regions. In this study, this was performed using
genome-wide SNP genotyping (by Dr Barry Chioza and Joe Leslie, University of
Exeter) using the Illumina CytoSNP-12v2.1 array and following the Infinium® HD
Assay Ultra manual protocol. This protocol is performed over 3 days with 200 ng of
DNA at a concentration of 50 ng/μl per sample.
Day 1: DNA samples were denatured using a buffer containing 0.1 N sodium
hydroxide, and then neutralised. Samples were amplified during an overnight
incubation at 37°C.
Day 2: Amplified DNA samples were enzymatically fragmented using the Illumina
fragmentation solution (FMS), using endpoint fragmentation to avoid
overfragmentation. The DNA was then precipitated using 100% 2-propanol and the
Illumina precipitation solution (PM1), and collected via a 20 min centrifugation
performed at 4°C. The DNA was then resuspended using the Illumina resuspension,
hybridisation and wash solution (RA1), and then denatured at 95°C for 20 mins. The
denatured samples were cooled at room temperature for 30 mins before being loaded
onto the BeadChips (with each chip holding 12 samples). This was then incubated in
the Illumina hybridisation oven at 48°C for 16-24 hours.
59
Day 3: Beadchips were prepared for the staining process by washing with the Illumina
Prepare BeadChip Buffer 1 (PB1) solution in order to remove unhybridised and
nonspecifically hybridised DNA samples from the BeadChips. Labelled nucleotides
were dispensed onto the BeadChip through the flow-through chambers to perform
single-base extension of primers hybridised to the DNA samples. The BeadChips were
then stained using the Illumina XStain HD BeadChip process and imaged on an
Illumina iScan System. The iScan system uses a laser to excite the fluorophores of
the single-base extension product on the beads of the BeadChip. Light emissions from
the fluorophores were recorded by the reader, allowing high resolution images of the
BeadChip to be taken. The data from these images was analysed using the Illumina
GenomeStudio Integrated Informatics Platform, allowing the genotype to be
determined.
Further analysis was undertaken using the Illumina KaryoStudio v1.4 software for
identification of cytogenetic or chromosomal structural aberrations. The generated
data was also exported into Microsoft Excel 2013, and analysed visually, as well as
using an Excel macro written by Dr Barry Chioza, to highlight notable genomic regions
(>1 Mb) with a shared haplotype.
2.3.5 Next generation sequencing (NGS)
TruSightTM One Sequencing Panel
The Illumina TruSightTM One clinical exome sequencing panel was performed by Luke
O’Gorman and Chelsea Norman, University of Southampton. This panel provides
targeted sequencing for 4813 genes associated with clinical phenotypes, including a
number of known causative genes for inherited ocular disease. Next generation
sequencing was performed using the Illumina TruSightTM capture kit and the NextSeq
500 platform (Illumina), with an approximate read depth and coverage of 20X minimum
for > 95% of the genome.
60
Whole exome sequencing (WES)
WES was predominantly performed by either BGI Tech Solutions (BGI Genomics,
Hong Kong) using a BGISEQ-500 platform or by the Exeter Sequencing Service
(University of Exeter, UK) using a Illumina NextSeq 500 platform, with an approximate
read depth and coverage of 20X minimum for 90-95% of the genome achieved with
both platforms.
Bioinformatic pipeline
Alignment of NGS data to the reference human genome sequence
Raw sequence data in FASTQ format was aligned to the Genome Reference
Consortium human genome build 37 (GRCh37) using the BWA-MEM (v0.7.12) (113)
alignment algorithm, generating output files in Sequence Alignment/Map (SAM)
format, which were then converted into Binary Alignment/Map (BAM) format using
SamFormatConverter (Picard Tools v2.15.0) for further analysis. FixMateInformation
(Picard Tools v2.15.0) was used to verify read pair information between each read and
its corresponding mate pair and fix information errors if necessary, sorting the output
file by read coordinates. Duplicate reads were removed using MarkDuplicates (Picard
Tools v2.15.0). This identifies the duplicate reads by matching all read pairs with
identical 5’ coordinates and orientations, avoiding bias in variant calling by presenting
variants with artificially high read depth support. Indel (insertion and deletion)
realignment and base quality recalibration was performed using the Genome Analysis
Toolkit (GATK) v3.70 (114).
Identification of sequence variation
Variant calling for SNPs and short indels was performed using GATK HaplotypeCaller
(v3.70) with a variant call format (VCF) file generated for each individual. This was
filtered for quality control based on read depth (DP), mapping quality (MQ), strand
bias, and relative position of the variant within the read.
Copy number variants (CNV) (WES data only) were detected using ExomeDepth
(115), which uses read depth data to call CNVs from exome sequencing experiments,
and SavvyCNV (116), which calls CNVs using off-target read data.
61
Annotation of sequence variation
Functional annotation of variants in the filtered VCF file was then performed using
Alamut® Batch (v1.10), facilitating interpretation of variants in a clinical context. Each
variant was annotated on all available transcripts.
Variant prioritisation
Variants (SNPs and short indels) were prioritised using the following criteria (Table
2.4).
Table 2.4 Criteria used for variant prioritisation
Criteria Description
Call quality • VCF filter = PASS
• MQ ≥ 50
• DP ≥ 5
Integrative Genome Viewer (IGV) was also used to
visualise the aligned sequencing data and genomic region
adjacent to the variant
Consistent with mode
of inheritance
If pedigree suggests:
• Autosomal recessive inheritance: homozygous and
compound heterozygous variants
• Autosomal dominant inheritance: heterozygous
variants
• X-linked inheritance: hemizygous variants (in males)
Frequency in control
datasets
MAF <0.01 in:
• gnomAD (v2.1.1 and v3.1.1) (all populations)
• Relevant internal control exome dataset of unrelated
Amish (220) or Pakistani (65) individuals
Predicted impact on
protein or splicing
• Non-synonymous missense variants
• Presumed loss of function variants
• Splicing variants, defined as intronic variants within
5bp of the intron-exon junction
In silico pathogenicity
predictions
Non-synonymous missense variants:
• SIFT score < 0.05
• PolyPhen-2 score > 0.15
• PROVEAN score < -2.5
Splicing variants:
62
• Local splicing effect predictions calculated using the
Alamut® Batch interpretation algorithm that interprets
splice site signals recognised by MaxEntScan,
SpliceSiteFinder-like (SSF) and Splice Site Prediction
by Neural Network (NNSplice)
Previously reported
variants
• ‘Pathogenic’ and ‘Likely pathogenic’ annotation in
ClinVar
• ‘Disease-causing mutation’ and ‘Disease-causing
mutation?’ annotation in HGMD professional v2020.4
For glossary of terms see Appendix A.
2.4 Literature review
Literature reviews were performed using Pubmed (https://pubmed.ncbi.nlm.nih.gov/),
Google Scholar (https://scholar.google.com/) and HGMD® Professional 2020.4
(https://my.qiagendigitalinsights.com/bbp/view/hgmd/pro/start.php) to retrieve all
reported disease-associated loci and variants. Additional information collated where
relevant included the number of families and number of affected individuals within
each family, their country of origin, and reported phenotype. The variants were then
searched for within NCBI ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/) to identify any
additional literature reports and evidence supporting causality.
63
3 STUDIES OF OCULOCUTANEOUS ALBINISM (OCA) IN
COMMUNITIES
3.1 Introduction
OCA refers to a group of genetically and clinically heterogeneous disorders
characterised by abnormal melanin synthesis, resulting in decreased or absent
pigmentation of eyes, skin and hair. Six genes involved in the melanin biosynthesis
pathway are known to cause non-syndromic OCA, with gene variants in TYR, OCA2,
TYRP1, SLC45A2, SLC24A5 and C10orf11 associated with OCA subtypes 1, 2, 3, 4,
6 and 7 respectively. OCA5 has been mapped to the chromosome 4q24 locus,
although the gene responsible has not yet been identified (117). OCA may also be a
part of a broader systemic phenotype in conditions associated with lysosomal storage
defects such as Hermansky-Pudlak syndrome (associated with pathogenic variants in
HPS1, HPS3, HPS4, HPS5, HPS6, AP3B1, DTNBP1, BLOC1S3, BLOC1S6 and
AP3D1) and Chediak-Higashi syndrome (associated with pathogenic variants in
LYST). Both syndromic and non-syndromic forms of OCA are inherited as autosomal
recessive conditions.
Ocular features are present in individuals with OCA, and are characteristic of the
disease. These include photophobia, nystagmus, foveal hypoplasia, iris
transillumination and abnormal decussation of nerve fibres at the optic chiasm
resulting in crossed asymmetry on visual evoked potential testing (118). These ocular
features may however be variable with no single defining characteristic found to be
present in every individual with OCA (119). The cutaneous phenotype may also vary,
ranging from total absence to near normal levels of pigmentation, and can be difficult
to evaluate, particularly in individuals with a lightly pigmented ethnic background (120,
121). As such, OCA can be difficult to distinguish clinically from several other ocular
disorders with overlapping phenotypical features, such as GPR143-associated X-
linked ocular albinism, where the hypopigmentation is limited to the eye (122),
FRMD7-associated X-linked idiopathic congenital nystagmus (123), SLC38A8-
associated foveal hypoplasia (also known as FHONDA; foveal hypoplasia, optic nerve
64
decussation defects, and anterior segment dysgenesis) (124), and dominant PAX6-
related ocular developmental disorders (125).
OCA1, associated with TYR gene variants, is the most common OCA subtype found
in Caucasians accounting for ~50% of cases worldwide (126, 127). TYR encodes the
enzyme tyrosinase, which is the critical and rate limiting enzyme in the biosynthesis of
melanin in follicular and epidermal melanocytes in hair and skin, as well as in uveal
melanocytes in the iris, ciliary body and choroid, and RPE cells in the eye (128).
Disease-associated variants in the TYR gene cause complete or partial OCA1
depending on their impact on the residual activity of the encoded mutant tyrosinase
enzyme (129). TYR gene variants that result in a severe reduction or complete
abolition of enzyme activity are associated with OCA1A, characterised by an almost
complete absence of hair, skin and eye pigmentation (129, 130). Hypomorphic TYR
variants in which mutant tyrosinase possess residual catalytic activity are associated
with OCA1B, where affected individuals present with a milder phenotype with reduced
levels of pigmentation (129, 130).
This chapter entails clinical and genomic investigations into the cause of OCA in four
interlinking Amish families, which alongside functional studies and a review of
previously published OCA genomic studies, highlights the pathogenicity of two
common hypomorphic TYR variants p.(Ser192Tyr) and p.(Arg402Gln) that were
previously largely considered non-pathogenic polymorphisms. Additionally, this
chapter compiles genetic findings regarding the causes of OCA in 36 families from
several communities in Pakistan. Together with a comprehensive literature review of
all pathogenic gene variants associated with OCA in the Pakistani population, these
findings permit a detailed understanding of the molecular spectrum of OCA in
Pakistan.
Within this chapter, I was responsible for the interpretation and analysis of all collected
clinical data for all affected individuals, including the development of clinical proformas
to aid collaborating scientists acquire the best quality and most relevant clinical data
for the Pakistan OCA families (families 5 - 40) described in chapter 3.3. I performed
DNA extraction for a proportion of affected families (remaining DNA extraction largely
completed by Joe Leslie, University of Exeter), primer design for amplification of TYR
65
exons 4 and 5 (primers for amplification of TYR exons 1-3 designed by Dr Gaurav
Harlalka, University of Exeter) as well as for all variants identified in families 5 – 39,
and completed all cosegregation studies. I was also responsible for analysing results
of all exome sequencing and SNP mapping studies performed in this chapter. All
literature reviews within this chapter were performed by myself, including the statistical
analysis across different OCA cohorts in chapter 3.2, as well as the compilation of all
pathogenic gene variants associated with OCA in the Pakistani population, detailed in
appendix C.
66
3.2 Evidence that the Ser192Tyr/Arg402Gln in cis Tyrosinase gene
haplotype is a disease-causing allele in oculocutaneous albinism
type 1B (OCA1B)
3.2.1 Introduction
The apparent missing heritability in OCA is well described, with ~25-30% of clinically
affected individuals lacking two clearly pathogenic sequence alterations within the
same OCA gene; this proportion is higher in individuals with a partial OCA phenotype
(129, 131). Several hypotheses have been proposed to explain this missing
heritability, including variants in promoter or other regulatory elements, as well as
epistatic or synergistic interactions between known genes (129, 132). Two TYR
sequence variants [NM_000372.4:c.575C>A; p.(Ser192Tyr) or S192Y and
c.1205G>A; p.(Arg402Gln) or R402Q], previously described as non-pathogenic
polymorphisms due to their frequency in the general population (25% and 18%
respectively), have been found to be enriched in cohorts of OCA patients with only
one identified TYR mutation (126, 129, 133-141), leading to suggestions that these
variants may in fact account for some of this missing heritability (117, 126, 127, 133,
134, 137, 142-145). This has however been disputed by others (136, 138, 146) who
have hypothesised that these variants may be pathogenic only when present in cis,
and inherited in biallelic fashion with a second deleterious TYR variant for tyrosinase
activity to be sufficiently reduced to a level that will cause an OCA phenotype (132,
145, 147). This hypothesis is supported by increasing numbers of individuals with OCA
reported to carry one pathogenic variant as well as the p.(Ser192Tyr) and
p.(Arg402Gln) variants in TYR (132, 141, 148, 149). However, due to the high
frequency of the p.(Ser192Tyr) and p.(Arg402Gln) variants in the general population,
and the often small family sizes common to modern European populations, in many
cases it has not been possible to obtain informative allele segregation to phase gene
variants and prove inheritance of a cis p.(Ser192Tyr)/p.(Arg402Gln) haplotype in trans
with the pathogenic TYR alteration in all affected individuals (145, 148). The remaining
uncertainty in clinical interpretation of this haplotype limits its routine reporting in
diagnostic gene panels. This has important diagnostic implications; designating the
TYR p.(Ser192Tyr)/p.(Arg402Gln) haplotype as pathogenic could substantially
67
increase the diagnostic yield by ~25-50% in albinism patient cohorts with missing
heritability (132). This also further supports the hypothesis that the prevalence of
OCA1, commonly quoted as ~1 in 40,000 (130), likely represents a substantial
underestimation particularly amongst Caucasian populations with fair pigmentation
(149).
This study entails extensive genetic studies stemming from the investigation of a large
multigenerational extended Amish family, alongside functional studies, and a review
of genotyped UK based albinism cohorts and of existing literature, to provide strong
evidence supporting pathogenicity of the TYR p.(Ser192Tyr)/(Arg402Gln) in cis
haplotype and its contribution to OCA1B in European populations.
3.2.2 Materials and methods
Amish patient ascertainment and molecular genetic analysis
Affected individuals and unaffected family members from four Ohio and Wisconsin
Amish families with a common Ohio ancestry were recruited to this study with informed
consent (Figure 3.1). A medical history was taken in all recruited family members, as
well as detailed phenotyping of skin and hair pigmentation, particularly in the context
of familial pigmentary background. A diagnosis of nystagmus was established in all
affected individuals (apart from individual X:5), and further ophthalmic investigations
including electroretinography and OCT were performed in selected individuals. A
diagnosis of albinism was made following a set of diagnostic criteria proposed by Kruijt
et al (119).
Blood/buccal samples were obtained with informed consent for DNA extraction
(section 2.3.2). Exome sequencing (WES for individual IX:9 and Illumina TruSightTM
One clinical exome sequencing panel for individual IX:22) was performed as described
in section 2.3.5. Bioinformatic analysis of exome data was performed as per section
2.3.5, with additional filtering performed using the “Albinism or congenital nystagmus
v1.0” PanelApp virtual gene panel (41 genes)
(https://panelapp.genomicsengland.co.uk/panels/).
68
Primers were designed as described in section 2.3.3 to cover all coding exons and
associated intron-exon junctions of TYR (Appendix Table D1). As the 3’ region
encompassing coding exons 4 and 5 of TYR shares high homology with a pseudogene
(TYRL) (150), locus-specific amplification primers were designed for TYR exons 4 and
5 to prevent co-amplification of TYR and TYRL and subsequent misinterpretation of
results. PCR and dideoxy sequencing was performed as described in section 2.3.3 to
genotype and confirm appropriate segregation of candidate disease variants in all
available affected and unaffected individuals.
Establishment of Tyr mutant cell lines, cell culture conditions and enzymatic activity
assays
(Performed by Chelsea Norman and Aida Sanchez-Bretaño, University of
Southampton)
The plasmid vector p3XFLAG-CMV-14 containing TYR cDNA was purchased from
Addgene (Massachusetts, USA) and was initially deposited by Ruth Halaban (151).
Upon arrival, sequencing revealed the p.(Ser192Tyr) (c.C575A) common population
variant to be present. Site-directed mutagenesis was used to create the wild-type
sequence (c.575C, p.192Ser) as well as the p.(Arg402Gln) variant. Site-directed
mutagenesis was carried out using the non-strand displacing activity of Pfu DNA
polymerase to incorporate and extend the mutagenic primers. The reaction mixture
contained Phusion Pfu Polymerase and its buffer, forward and reverse primers (0.5
µM), dNTPs (200 µM), and the cDNA template. PCRs were performed in a total volume
of 50 µl. Touch-down PCR conditions were set at 98°C for 30 sec followed by 30 cycles
of 98°C for 10 sec, 45-72°C for 10 - 30 sec and 72°C for 15-30 sec, and a final
extension step of 72°C for 5 - 10 min. The PCR product was treated with DpnI to digest
the methylated parental DNA.
Purified mutated tyrosinase PCR products were employed to transform NEB® 5-alpha
Competent E. coli (High Efficiency; New England Biolabs, UK) via heat shock method.
Briefly, 50 µl of thawed cells were kept on ice and combined with approximately 100
ng of plasmid DNA and incubated for 30 min. The cell-DNA mixture was heat shocked
at 42˚C for 30 sec and then placed on ice for 5 min. Cells were given S.O.C medium
and incubated for an hour in a shaking incubator before being plated on ampicillin
69
selection (100 ug/ml) LB agar plates. After overnight incubation at 37˚C, single
ampicillin resistant colonies were picked and grown in LB broth for approximately 16
hours, at which point the cells were pelleted by centrifugation and the DNA extracted.
When the stocks were diminished, competent cells were produced through treatment
with CaCl2 and subsequently transformed using the heat shock method described
above.
Human Embryonic Kidney 293 Freestyle (HEK293F) cells (Invitrogen, California, USA)
were cultured in Freestyle culture medium (Invitrogen, California, USA) at 37˚C in a
shaking incubator at 125 rpm with 8% CO2. When cells reached a density of 1 x106
cells/ml, they were transfected with 30 µg of plasmids containing the p.(Arg402Gln) or
p.(Ser192Tyr) mutations or co-transfected with both plasmids. The lipid-based
reagent, 293fectin (60 µl) (ThermoFisher, UK), was diluted in Opti-MEM
(ThermoFisher, UK) and incubated at room temperature for 5 min. DNA and 293fectin
were combined, gently mixed and incubated at room temperature for 30 min before
adding to cells. Then, cells were incubated in 6-well plates for 72 hours at 31°C or
37°C to reach 90% confluency, and the enzymatic activity assays were performed.
DOPA-oxidase activity was assessed in the different mutants. First, transfected cells
from the different mutant clones were treated with L-DOPA, and the DOPA-oxidase
activity was measured as the accumulation of the downstream product, dopachrome,
following the manufacturer’s protocol. Briefly, cells cultured in 6-well plates were lysed
in NP40 Cell Lysis Buffer (ThermoFisher, UK) containing 1 mM phenylmethylsulfonyl
fluoride (PMSF) (in DMSO with a final concentration of 1%) and 1X protease and
phosphatase inhibitor (Halt™ Phosphatase Inhibitor Cocktail, Thermo Fisher
Scientific, UK), and protein concentration was measured by BCA assay (Thermo
Scientific™ Pierce™ BCA Protein Assay Kit). Samples were then diluted into 4 µg/µl,
and 50 µl or 30 µl sample aliquots were used for the DOPA assays. After adding the
volume of the samples to 96-well plates, 150 µl of a phosphate buffer with L-DOPA 1
mM was added to the wells. Enzymatic activity was recorded as the absorbance of
dopaquinone at 492 nm from the start of L-dopa treatment (0 min) and at 30 min
intervals thereafter for a total of 180 min at both 31°C and 37°C. Assays were routinely
performed in triplicate and the results are presented as the means of the independent
assays +/- standard error.
70
Results of enzymatic activity at 180 min were normalized to wild type, with the values
for wild type taken to be 100% of the expected enzymatic activity. One-way ANOVA
was performed followed by a Sidak’s post-hoc test. A probability level of at least
p<0.05 was considered statistically significant (* p<0.05, ** p<0.01, *** p<0.001, ****
p<0.0001).
Evaluating the prevalence of TYR p.(Ser192Tyr)/p.(Arg402Gln) haplotype in OCA and
control cohorts
A clinical cohort of affected individuals with nystagmus and/or albinism was
retrospectively ascertained through the Southampton (161 individuals) and Salisbury
(131 individuals) research databases. All individuals had been referred from a regional
paediatric nystagmus clinic and recruited with informed consent. NGS (Illumina
TruSightTM One clinical exome sequencing panel), alignment and filtering was
performed as previously described in section 2.3.5. The genomic data were
interrogated to ascertain frequency of the TYR p.(Ser192Tyr)/p.(Arg402Gln) haplotype
in this cohort. A literature review was also performed to evaluate the reported
prevalence of the TYR p.(Ser192Tyr)/p.(Arg402Gln) haplotype in additional published
OCA cohorts. This was compared against an in-house exome database of 219 Amish
individuals unaffected by OCA. Statistical analysis was performed using an
established software package (R Core Team 2015; R Foundation for Statistical
Computing, Vienna, Austria) (152).
3.2.3 Results Clinical and genetic findings
A large multigenerational extended Amish family residing in Wisconsin (USA) with nine
affected individuals all exhibiting nystagmus and variable levels of hair and skin
hypopigmentation was investigated (Figure 3.1A; family 4). On the basis of a detailed
medical history, assessment of skin and hair pigmentation, and ophthalmic
investigations in selected affected individuals, a diagnosis of likely mild OCA was
made in all affected individuals. Two additional Amish families with a total of four
71
affected individuals with a similar clinical phenotype were subsequently recruited
(Figure 3.1A; families 2 and 3). In addition, a further Amish family with a single affected
individual with OCA was recruited to the study (Figure 3.1A; family 4). This individual
displayed clinical features consistent with a complete OCA phenotype, including pale
skin and white/blonde hair and eyelashes, nystagmus, iris transillumination defects
and foveal hypoplasia. Affected individuals were not noted to bruise or bleed easily,
although specific haematological investigations were not performed. Clinical findings
for all affected individuals are summarised in Table 3.1.
Table 3.1 Summary of clinical features observed in affected individuals in families 1 - 4 with OCA
Family (ID) Nystagmus Hair colour
Eye colour
Other ocular features Other systemic features
1 (X:1) ✓ Blonde Blue Iris transillumination defects,
depigmented fundus, foveal
hypoplasia, alternating
esotropia, optic disc hypoplasia
-
2 (X:2) ✓ NA NA Blunted foveal reflex,
depigmented fundus
NA
3 (X:3) ✓ Blonde Blue Iris transillumination defects,
blunted foveal reflex
-
3 (X:4) ✓ Dark
blonde
Blue transillumination defects, foveal
hypoplasia, strabismus
-
3 (X:5) NA Strawberry
blonde
NA Blunted foveal reflex -
4 (IX:9) ✓ Blonde Blue Pale fundi, iris transillumination
defects, foveal hypoplasia,
myopia, strabismus.
Nyctalopia, photosensitivity and
peripheral VF loss with normal
ERG
4 (IX:10) ✓ Pigmented Blue - Mild learning
difficulties
4 (IX:12) ✓ Light brown NA - -
4 (IX:14) ✓ Dark brown Blue Pale fundi -
4 (IX:15) ✓ Pigmented Blue - -
4 (IX:16) ✓ NA NA NA NA
4 (IX:20) ✓ Blonde Blue - -
4 (IX:22) ✓ White/
blonde
Blue - -
4 (X:15) ✓ Brown Brown Myopia Neonatal
intraventricular
haemorrhage
Abbreviations: ERG, electroretinogram; NA, information not available; VF, visual field. The (✓)
and (-) symbols indicate the presence of absence of a feature in an affected subject respectively
72
All affected individuals were diagnosed with hypomorphic albinism on the basis of a positive molecular diagnosis as well as the presence of either one major criterion or two minor criteria described by Kruijt et al (119). Major criteria includes foveal hypoplasia ≥ grade 2, crossed asymmetry on visual evoked potential testing and ocular hypopigmentation (either iris transillumination or fundus hypopigmentation ≥ grade 2), whilst minor criteria includes nystagmus, hypopigmentation of skin and hair, and grade 1 fundus hypopigmentation and/or foveal hypoplasia.
Exome sequencing was initially performed in two affected individuals in family 4
(individuals IX:9 and IX:22) for targeted evaluation using the “Albinism or congenital
nystagmus v1.0” PanelApp virtual gene panel (41 genes). Subsequently, variants
predicted to have a functional consequence (including CNVs) located genome-wide
were identified and filtered according to allele frequency (gnomAD MAF of <0.01). This
identified only a single plausible candidate disease variant in both individuals, a
heterozygous TYR missense variant (GRCh38) chr11:g.89178708T>G;
NM_000372.4:c.755T>G; p.(Met252Arg) or M252R. The p.Met252 amino acid residue
is located in the catalytic domain of the tyrosinase protein, and is conserved across a
variety of vertebrate species (Figure 3.1B). This variant was absent in gnomAD and
Genome Project population databases, although it was present in an Amish control
exome dataset (allele frequency 0.0023) in heterozygous form only. In silico analysis
of the p.(Met252Arg) variant using SIFT, PolyPhen-2 and PROVEAN predicted the
variant to be deleterious, possibly damaging and deleterious. This variant has been
reported in compound heterozygous form [with a previously reported p.(Arg217Trp)
variant] in a single individual with OCA (141), and is considered to be likely pathogenic.
Exome sequencing did not identify any additional candidate single nucleotide or
structural disease variants in any OCA-associated genes.
To explore this apparent missing heritability, targeted dideoxy sequencing of all coding
regions and intron-exon junctions of the TYR gene was performed in these two
individuals. This confirmed the presence of the p.(Met252Arg) variant, and also
identified a further two TYR missense variants (GRCh38) chr11:g.89178528C>A;
NM_000372.4:c.575C>A; p.(Ser192Tyr) (S192Y) and (GRCh38)
chr11:g.89284793G>A; NM_000372.4:c.1205G>A; p.(Arg402Gln) (R402Q) in the
same two individuals, excluded from the exome sequencing analysis based on
population allele frequencies of 0.25 and 0.18 respectively. Segregation of all three
TYR variants in all Amish families (families 1-4) is shown in Figure 3.1A, which
73
demonstrates that the p.(Ser192Tyr)/p.(Arg402Gln) variants are linked in cis and
inherited in a compound heterozygous fashion with p.(Met252Arg) [which itself occurs
in cis with p.(Arg402Gln)] in all affected individuals except for a single affected
individual with OCA (family 1; individual X:1), found to be homozygous for
p.(Met252Arg) through targeted dideoxy sequencing. Individuals compound
heterozygous for TYR p.(Met252Arg) and p.(Ser192Tyr)/p.(Arg402Gln) alleles
displayed clinical features suggestive of partial albinism with variable skin and hair
depigmentation, whilst the individual homozygous for the TYR p.(Met252Arg) variant
displayed features of classical OCA including nystagmus, iris transillumination defects,
a depigmented fundus and foveal hypoplasia (Table 3.1). Notably, individuals carrying
the TYR p.(Met252Arg) variant on one allele and only the p.(Arg402Gln) or the
p.(Ser192Tyr) variant on the other allele were apparently unaffected with no clinical
features of OCA (individuals VIII:9, IX:2, IX:21, X:6, X:8, IX:1 and IX:4, Figure 3.1A).
Additive temperature sensitive effects of p.(Ser192Tyr) (S192Y) and p.(Arg402Gln)
(R402Q) variants on TYR enzymatic activity
The TYR p.(Arg402Gln) variant alone has previously been proposed to contribute to
OCA when inherited in trans with a pathogenic TYR variant (127, 133-135, 141-144,
153). Pedigree analysis however appears to dispute this, with five individuals
compound heterozygous for the pathogenic TYR p.(Met252Arg) variant as well as the
p.(Arg402Gln) variant and yet showing no clinical features of OCA (individuals VIII:9,
IX:2, IX:21, X:6 and X:8; Figure 3.1A). At the same time, 13 individuals who were
compound heterozygous for TYR p.(Met252Arg) and p.(Ser192Tyr)/p.(Arg402Gln)
alleles all displayed clinical features of partial albinism, suggesting an additive impact
of the p.(Ser192Tyr) and p.(Arg402Gln) variants on tyrosinase function. To investigate
this further, functional experiments were designed to study and quantify the effects of
the p.(Ser192Tyr) and p.(Arg402Gln) variants both independently and in combination,
compared to wild-type tyrosinase enzyme.
Figure 3.1C shows the DOPA-oxidase activity for all tyrosinase mutants analysed from
0 mins to 180 mins at 31°C and 37°C. At 37°C, a slight decrease in DOPA-oxidase
activity of the p.(Ser192Tyr) mutants, and an almost total loss of DOPA-oxidase
74
activity in the p.(Arg402Gln) mutants and p.(Ser192Tyr)/p.(Arg402Gln) double
mutants was observed. Tyrosinase activity recorded at 31°C was more variable across
the different mutant groups, with significant decrease in activity in all TYR-mutant cell
lines compared to wild type. For all the TYR-mutant cell lines, the
p.(Ser192Tyr)/p.(Arg402Gln) double mutants showed the most reduced tyrosinase
activity, followed by p.(Arg402Gln) mutant, with the p.(Ser192Tyr) mutant least
affected. There was a statistically significant difference between all three mutant
groups, indicative of a cumulative effect of both p.(Ser192Tyr) and p.(Arg402Gln)
mutations on tyrosinase activity.
75
Figure 3.1 Pedigree diagrams, TYR genotype and functional data
(A) Pedigree diagram of families 1 - 4 showing segregation of the TYR p.(Ser192Tyr), p.(Met252Arg), and p.(Arg402Gln) variants. The two disease-causing haplotypes are shaded; the p.(Met252Arg) haplotype in blue, and the p.(Ser192Tyr)/p.(Arg402Gln) in cis haplotype in yellow, with the relevant amino acid change highlighted in red. All affected individuals inherited both the p.(Met252Arg) and p.(Ser192Tyr)/p.(Arg402Gln) in cis haplotypes in compound heterozygous fashion, apart from individual X:1, who displayed a more severe albinism phenotype and was found to be homozygous for the p.(Met252Arg) variant. No unaffected individuals inherited both disease-associated haplotypes, confirming segregation with disease in the family. (B)(i) Sequence chromatograms showing the TYR c.575C>A; p.(Ser192Tyr), c.755T>G; p.(Met252Arg) and c.1205G>A; p.(Arg402Gln) variants in heterozygous form (ii) Schematic localisation of TYR p.(Ser192Tyr), p.(Met252Arg) and p.(Arg402Gln) variants within the
76
catalytic tyrosinase domain of the TYR polypeptide. The p.(Ser192Tyr) and p.(Arg402Gln) variants are located at or near the copper-containing catalytic binding sites (the red diamonds denote the histidine residues that bind to copper atoms and hence structurally coordinate the positions of the metal binding sites) (iii) Conservation of the TYR p.(Ser192Tyr), p.(Met252Arg) and p.(Arg402Gln) variants across species. (C) Tyrosinase activity in wild-type, p.Ser192Tyr (S192Y) mutant, p.Arg402Gln (R402Q) mutant and double mutant HEK293 cells. Absorbance of dopaquinone, a product synthesised by the transformation of L-DOPA by tyrosinase was quantified as a measure of tyrosinase activity in wild-type and TYR-mutant cell lines. Cumulative production of dopaquinone (i, ii)) was quantified from the start of the L-DOPA treatment (0 min) to 180 min. Statistical differences between cell lines were analysed at 180 min (iii, iv). Data is shown as mean ± SEM and statistically significant differences between groups are indicated by asterisks (* p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001); ns = not significant
Enrichment of the TYR p.(Ser192Tyr)/p.(Arg402Gln) haplotype in OCA and control
cohorts
Interrogation of a clinical cohort of 161 affected individuals with nystagmus and/or
albinism (Southampton cohort) [including individuals previously reported by Norman
et al and O’Gorman et al (132, 145)] identified 71 individuals with two pathogenic or
likely pathogenic variants (molecularly diagnosed including TYR, OCA2, GPR143 and
PAX6 genes), 51 individuals carrying only a single likely disease-associated TYR
variant with no candidate pathogenic variants identified in other OCA genes (missing
heritability), and 39 individuals with no disease-associated TYR variants. All patients
were sequenced using either using the “Albinism or congenital nystagmus v1.0”
PanelApp gene panel (41 genes) (https://panelapp.genomicsengland.co.uk/panels/)
or a broader research panel as previously described (132, 145). CNV analysis was not
performed. Of these, 2 of the 71 individuals in the molecularly diagnosed group and
49 of the 51 individuals in the missing heritability group were found to have a genotype
consistent with the presence of the TYR p.(Ser192Tyr)/p.(Arg402Gln) haplotype (i.e.
individuals who were homozygous or heterozygous for both these variants) (Table
3.2); this information was unavailable for the 39 molecularly undiagnosed individuals
in this clinical cohort.
A review of seven published OCA cohorts with missing heritability (i.e. individuals in
whom only a single pathogenic TYR variant has been identified), together with our
study cohort, found that approximately half of all affected individuals (50.7%) had a
genotype consistent with the TYR p.(Ser192Tyr)/p.(Arg402Gln) haplotype (Table 3.2).
This is markedly enriched compared to molecularly diagnosed TYR OCA cohorts
77
(2.0%), as well a control cohort of 219 Amish individuals with no OCA diagnoses
(31/219 or 16.9%; Pearson’s Chi-squared test, p < 2.2 x 10-16 ). These findings strongly
suggest that the TYR p.(Ser192Tyr)/p.(Arg402Gln) haplotype contributes to the OCA
phenotype.
Additionally, 49 affected individuals in the Southampton and Salisbury missing
heritability study cohorts identified as carrying only a single pathogenic or likely
pathogenic TYR variant were also found to harbour homozygous or heterozygous TYR
p.(Ser192Tyr) and p.(Arg402Gln) variants; of these, familial segregation was
performed in 41 individuals and their parents to assess the phase. In 23 individuals,
this confirmed that the TYR p.(Ser192Tyr) and p.(Arg402Gln) variants were inherited
in cis, and this haplotype was in trans to the previously identified pathogenic or likely
pathogenic TYR variant (Table 3.3). For the remaining 18 cases, definitive segregation
was not possible. Notably, no case was identified in which segregation showed that
p.(Ser192Tyr) and p.(Arg402Gln) were not in trans with the pathogenic or likely
pathogenic variant.
In five of the seven published OCA cohorts with missing heritability reviewed, it was
possible to determine the cis/trans phase of the TYR p.(Ser192Tyr) and p.(Arg402Gln)
variants in a proportion of individuals reported (136, 139, 141, 148, 149) (Table 3.3);
in the remaining individuals this was not possible due to familial samples being
unavailable for segregation analysis, or uninformative segregation results (owing to
the high allele frequency of the p.(Ser192Tyr) and p.(Arg402Gln) TYR variants in the
general population). For the remaining two studies of OCA cohorts with missing
heritability, the cis/trans phase of the TYR p.(Ser192Tyr) and p.(Arg402Gln) variants
could not be determined from the reported genotypes (126, 134). There were 41 OCA
individuals with missing heritability from these five studies in whom the
p.(Ser192Tyr)/p.(Arg402Gln) haplotype was possible, and where the cis/trans phase
of the TYR p.(Ser192Tyr) and p.(Arg402Gln) variants could also be determined. In
accordance with the findings from our local research cohorts, together with this
additional informative cohort derived from five published studies, the TYR
p.(Ser192Tyr)/p.(Arg402Gln) haplotype segregated in trans with the pathogenic TYR
variant in all cases (amounting to 25.5% of total missing heritability cases; Table 3.3).
Taken together with the findings in Table 3.2, this suggests that the
78
p.(Ser192Tyr)/p.(Arg402Gln) haplotype completes the molecular diagnosis in ~25-
50% of OCA individuals with missing heritability.
79
Table 3.2 Prevalence of TYR p.(Ser192Tyr)/S192Y and p.(Arg402Gln)/R402Q variants in OCA cohorts
OCA cohorts with missing heritability
(individuals with only 1 TYR pathogenic or likely pathogenic variant identified)
Molecularly diagnosed OCA1 cohorts
(individuals with 2 TYR pathogenic or likely pathogenic variant
identified)
This study* Hutton &
Spritz 2008a
Hutton & Spritz
2008b
Oetting
2009
Ghodsinejad
Kalahroudi
2014
Lasseaux
2018
Gronskov 2019 Campbell 2019 Hutton & Spritz
2008b
Oetting 2009 Ghodsinejad
Kalahroudi
2014
Gronskov 2019
Phenotype Nystagmus
and/or
albinism
AROA/ mild
OCA
OCA OCA1 OCA1 Nystagmus
and/or
absence of
fovea
Albinism (OCA,
AROA or OA)
Nystagmus and at
least one other
ocular feature of
albinism, no skin
hypopigmentation
OCA OCA1 OCA1 Albinism (OCA,
AROA or OA)
Country
(ethnicity)
England (Caucasian) USA, Canada,
Northern Europe
(non-Hispanic/
Latino
Caucasians)
NA (Iranian) France Scandinavia
(Scandinavian)
England USA, Canada,
Northern Europe
(non-Hispanic/
Latino
Caucasians)
NA (Iranian) Scandinavia
(Scandinavian)
No of individuals
in cohort
51 20 13 3 6 158 29 4 71 9 19 2
No of individuals
hom or het for
both TYR S192Y
& R402Q
49 1 3 2 0 64 21 4 2 0 0 0
Proportion of
study cohort
where
S192Y/R402Q
haplotype is
possible
49/51
(96.1%)
1/20
(5.0%)
3/13 (23.1%) 2/3
(66.7%)
0/6
(0%)
64/158
(40.5%)
21/29
(72.4%)
4/4
(100%)
2/71
(2.8%)
0/9 (0%) 0/19
(0%)
0/2
(0%)
Combined
proportion where
S192Y/R402Q
haplotype is
possible
144/284
(50.7%)
2/101
(2.0%)
*This cohort includes individuals previously reported in Norman et al and O’Gorman et al Abbreviations: AROA, autosomal recessive ocular albinism; het, heterozygous; hom, homozygous; OCA, oculocutaneous albinism; OA, ocular albinism; no, number
80
Table 3.3 Potential contribution of TYR p.(Ser192Tyr)/p.(Arg402Gln) haplotype to molecular diagnoses in OCA cohorts with missing heritability This includes individuals with only 1 TYR pathogenic or likely pathogenic variant identified
Study This study* Oetting 2009 Ghodsinejad
Kalahroudi 2014
Lasseaux 2018 Gronskov 2019 Campbell 2019
Phenotype Nystagmus
and/or
albinism
OCA1 OCA1 Nystagmus and/or
absence of fovea
Albinism (OCA,
AROA or OA)
Nystagmus and at
least one other
ocular feature of
albinism, no skin
hypopigmentation
Country (ethnicity) England NA (Iranian) France Scandinavia
(Scandinavian)
England
Number of individuals in cohort 51 3 6 158 29 4
Number of individuals hom or het for both TYR S192Y
and R402Q, where S192Y/R402Q haplotype is possible
49 2 0 64 21 4
Number of individuals in whom it was possible to
determine the phase of TYR S192Y, R402Q and
pathogenic or likely pathogenic variants (“informative
cohort”)
23 2 6 31 6 2
Number of individuals in whom TYR S192Y and R402Q
were in cis, and in trans to pathogenic or likely
pathogenic TYR variant in the informative cohort
23 2 0 31 6 2
Proportion of “informative cohort” where S192Y/R402Q
haplotype is possible and molecular diagnoses due to
TYR S192Y/R402Q haplotype in trans to pathogenic or
likely pathogenic TYR variant
23/23
(100%)
2/2
(100%)
S192Y/R402Q
haplotype not
possible in any
individuals in study
31/31
(100%)
6/6
(100%)
2/2
(100%)
Combined proportion of “informative cohort” where
molecular diagnoses is due to TYR S192Y/R402Q
haplotype in trans to pathogenic or likely pathogenic
TYR variant
64/64
(100%)
Proportion of total cohort where molecular diagnoses
due to TYR S192Y/R402Q haplotype in trans to
pathogenic or likely pathogenic TYR variant
23/51
(45.1%)
2/3
(66.7%)
0/6
(0%)
31/158
(19.6%)
6/29
(20.7%)
2/4
(50%)
Combined proportion of total cohort where molecular
diagnoses is due to TYR S192Y/R402Q haplotype in
trans to pathogenic or likely pathogenic TYR variant
64/251
(25.5%)
81
*This cohort includes individuals previously reported in Norman et al and O’Gorman et al Abbreviations: AROA, autosomal recessive ocular albinism; het, heterozygous; hom, homozygous; OCA, oculocutaneous albinism; OA, ocular albinism.
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3.2.4 Discussion
The pathogenicity of TYR p.(Ser192Tyr) and p.(Arg402Gln) variants and their
contribution to the OCA phenotype, either in isolation or when linked in cis, has
been heavily debated in many studies (117, 126, 127, 133, 134, 136-138, 142-
146). As such, these TYR variants are variably reported by clinical testing
laboratories and potentially excluded, even when shown to be in cis. Here
genomic and functional data, initiated by a search for the cause of OCA in a
number of Amish families, provide irrefutably strong evidence that the TYR
p.(Ser192Tyr) and p.(Arg402Gln) variants are pathogenic when in cis. The
increased frequency of the TYR p.(Met252Arg) variant in the Amish community,
likely due to founder effects and endogamy together with the large family sizes
typical within the community, permitted empowered co-segregation studies able
to determine the haplotype, phasing and inheritance of the common
p.(Ser192Tyr) and p.(Arg402Gln) TYR variants together with the p.(Met252Arg)
variant in a large number of related individuals for the first time.
A number of previous studies have shown that the TYR p.(Arg402Gln) variant
is present at increased frequency in OCA patients in whom only a single clearly
deleterious sequence alteration in TYR has been detected (126, 129, 133-141,
149), and is proposed to contribute to OCA when inherited in trans with a
pathogenic TYR variant (127, 133-135, 141-144, 153). This has been disputed
by Oetting et al, who describe 10 unaffected parents of OCA individuals who
carried a combination of both a TYR pathogenic variant as well as the
p.(Arg402Gln) variant in trans, and yet showed no clinical features of OCA
(136). Recently, however, molecular and phenotypic analysis of a large cohort
of 268 French OCA1 individuals suggests that compound heterozygosity for a
known pathogenic TYR variant and the hypomorphic p.(Arg402Gln) variant may
be associated with a variable but generally mild form of albinism that may
remain clinically undiagnosed, with the authors supporting consideration of
p.(Arg402Gln) as a mildly pathogenic, but definitely pathogenic, TYR variant
when associated in trans with another pathogenic variant (154). It should be
noted that the TYR p.(Arg402Gln) variant is not generally thought to be
sufficiently deleterious to cause OCA in homozygous form, with a homozygous
83
prevalence of 7.47% in population databases (non-Finnish European
population, gnomAD v2.1.1), as well as several studies that have reported on
individuals homozygous for the p.(Arg402Gln) with no clinical features of
albinism (136, 138, 155).
It has been proposed that a second TYR variant, p.(Ser192Tyr), acting in cis
with the p.(Arg402Gln) variant, may have an additive effect producing a greater
reduction in enzyme activity compared to each variant individually (145, 147),
although this too has been disputed (154). Our study demonstrates that
inheritance of either variant individually in compound heterozygous form with
the deleterious p.(Met252Arg) variant is insufficient to result in a clinically
significant OCA phenotype (individuals VIII:9, IX:1, IX:2, IX:4, X:6 and X:8;
Figure 3.1A), although we acknowledge that subtle asymptomatic clinical
findings such as mild iris transillumination or foveal hypoplasia were not
specifically excluded. The p.(Ser192Tyr) and p.(Arg402Gln) variants are
believed to have arisen independently on different ancestral haplotypes (156),
and although these variants individually in Caucasian populations are common
with allele frequencies of 36% and 27% respectively (gnomAD v2.1.1), their
combined presence in cis on a recombinant haplotype is relatively rare,
predicted to be between 1.1% to 1.9% in European populations (145, 147, 149).
This study, alongside other previous studies (126, 132, 136, 141, 145, 148,
149), provides strong support to show that the TYR
p.(Ser192Tyr)/p.(Arg402Gln) haplotype is enriched in Caucasian OCA cohorts
with missing heritability (Table 3.2), and contributes to an OCA1B diagnosis
when inherited in trans with a second deleterious TYR variant (Table 3.3),
particularly in individuals with lower pigmentary backgrounds, who may be more
susceptible to the damaging effects of hypomorphic variants (135, 157).
Given the number of apparently unaffected individuals homozygous for the
p.(Ser192Tyr)/p.(Arg402Gln) haplotype reported in the literature (147-149), the
penetrance of the p.(Ser192Tyr)/p.(Arg402Gln) haplotype might appear to be
incomplete, confounding the argument that it is a pathogenic allele. The
phenotype of OCA2 can be modified by variants in MC1R (158) or TYRP1
(159), and the phenotype of OCA3 by haploinsufficiency of OCA2 (160);
84
additionally, possible digenic inheritance in OCA involving combinations of
TYR, OCA2 and SLC45A2 variants has been proposed (161, 162). Similarly,
the apparent reduced penetrance of the p.(Ser192Tyr)/p.(Arg402Gln)
haplotype may relate to the modifying effects of sequence variants in genes
encoding other melanosomal proteins, although other genetic and molecular
studies would be required to confirm this. However, we propose that individuals
homozygous for the hypomorphic p.(Ser192Tyr)/p.(Arg402Gln) allele have
instead a consistent but mild phenotype which is easily missed by incomplete
phenotyping. In support of this, our studies identified five individuals with a
clinical diagnosis of ‘possible hypomorphic’ OCA who were homozygous for
TYR p.(Ser192Tyr)/p.(Arg402Gln), with no other known or likely TYR or other
OCA gene-associated variants identified (Table 3.4). All were noted to have
foveal hypoplasia on OCT investigation, but most had very mild, if any, other
OCA features. Additionally, an apparently unaffected relative in our study was
also identified as homozygous for TYR p.(Ser192Tyr)/p.(Arg402Gln). Despite
the absence of nystagmus or any other pigmentary phenotype in this unaffected
individual and visual acuities of 0.1 and 0.08 LogMAR (right and left eye
respectively), further detailed clinical investigation identified very mild iris
transillumination and significant foveal hypoplasia (Table 3.4 and Figure 3.2). A
review of all affected and apparently unaffected individuals homozygous for
both TYR p.(Ser192Tyr) and p.(Arg402Gln) variants in the literature and our
study cohorts identified 13 affected individuals in three studies, and foveal
hypoplasia as well as iris transillumination was documented in all these
individuals (148, 149) (Table 3.4). It therefore seems likely that individuals
homozygous for both common variants and thus the hypomorphic
p.(Ser192Tyr)/p.(Arg402Gln) allele, have such a mild phenotype that they can
easily go unidentified and unreported due to minimal effects on visual function
or clear features of albinism.
85
Figure 3.2 Foveal hypoplasia in individual homozygous for TYR p.(Ser192Tyr)/p.(Arg402Gln) haplotype
SD-OCT (Spectral domain optical coherence tomography; Spectralis‐OCT, Heidelberg Engineering, Heidelberg, Germany) image of right (A) and left (B) eyes showing grade 3 foveal hypoplasia in an apparently unaffected individual homozygous for both TYR p.(Ser192Tyr) and p.(Arg402Gln) variants [foveal hypoplasia graded following structural grading system based on OCT data proposed by Thomas et al (163)]
The TYR p.(Arg402Gln) variant is located near the copper-containing catalytic
binding site CuB, and functional studies have shown that this amino acid
alteration results in an enzyme with decreased thermal stability, disrupted
copper binding and reduced catalytic activity, thought to be mediated by
decreased protein stability resulting in increased retention of the mutant
tyrosinase protein as an unprocessed and misfolded glycoform in the
endoplasmic reticulum (ER) (147, 155, 164-169). The TYR p.(Ser192Tyr)
variant is located within the copper-containing catalytic binding site CuA, and
has been shown to reduce tyrosinase enzymatic activity and melanocyte
pigment production independent of the p.(Arg402Gln) variant (147, 170, 171).
Genome-wide association studies have identified associations with skin, hair
and eye pigmentation for both p.(Ser192Tyr) and p.(Arg402Gln) variants (172-
176), suggesting these variants have a role in normal pigmentary variation, and
that the double-variant p.(Ser192Tyr)/p.(Arg402Gln) haplotype appears to
86
show an additive effect on these pigmentary phenotypes compared to each
variant individually (147). It is difficult however from literature review alone to
quantify the functional effects of the p.(Ser192Tyr) and p.(Arg402Gln) variants,
both independently and in combination, compared to wild-type tyrosinase
enzyme. This issue arises from the historical use of the human TYR expression
construct pcTYR containing the p.(Ser192Tyr) variant to study the effects of
“wild-type” tyrosinase activity (155, 177). Computational approaches to TYR
functional activity, based on protein flexibility and dynamic properties, suggest
that the p.(Ser192Tyr) and p.(Arg402Gln) variants both result in a TYR protein
that is less stable and has reduced enzyme activity compared to a wild-type
molecule; the combined effect of having both changes together in a single TYR
molecule however has not been previously investigated (178). This study now
shows for the first time a thermosensitive additive decrease in enzymatic
function of the double-variant p.(Ser192Tyr)/p.(Arg402Gln) TYR protein
compared to each variant acting individually (Figure 3.1C), lending further
support to pathogenicity of the p.(Ser192Tyr)/p.(Arg402Gln) haplotype.
Homology modelling of tyrosinase protein structure does not appear to show a
direct interaction between the 192 and 402 amino acid residues (149), and
therefore this additional reduction in enzyme function in the double-mutant
protein may instead be mediated by a combination of increased ER retention of
the misfolded mutant protein [caused by p.(Arg402Gln) reducing protein
stability] and reduced enzyme activity of any released mutant protein [possibly
resulting from steric hindrance effects of p.(Ser192Tyr) affecting the CuA
binding site] (170), as proposed by Gronskov et al (149).
Subcellular localisation studies have determined that disease-associated TYR
variants commonly result in near absolute and irreversible ER retention of the
mutant protein. The p.(Arg402Gln) variant however results in a thermosensitive
tyrosinase protein that is retained in the ER at higher temperatures, but is able
to partially exit the ER at lower, more permissive temperatures (164, 167, 169).
Homozygosity for the p.(Ser192Tyr)/p.(Arg402Gln) haplotype may therefore
still permit sufficient quantities of mutant tyrosinase to reach the inner surface
of the melanosomal membrane, where the mutant protein is still able to
participate in protein-protein interactions with other melanosomal proteins
87
involved in melanogenesis, such as TYRP1 and TYRP2 (179, 180), resulting in
a less severe functional impact and a milder pigmentary phenotype that may
not always be clinically significant. This thermosensitivity of the double variant
mutant TYR protein also provides a compelling explanation for our novel
discovery of a consistent foveal hypoplasia phenotype in individuals who are
homozygous for both p.(Ser192Tyr)/p.(Arg402Gln) TYR variants, as higher
temperatures within the developing eye may result in a larger impact of these
variants on tyrosinase function (133), whilst lower temperatures at the skin and
extremities instead results in greater preservation of mutant protein function
and a milder and more variable pigmentary phenotype.
Together, our studies define the genotype, biochemical and phenotype
correlation of the p.(Met252Arg) and p.(Ser192Tyr)/p.(Arg402Gln) TYR
variants and collectively demonstrate that the in cis
p.(Ser192Tyr)/p.(Arg402Gln) allele is pathogenic. As such, the TYR
p.(Ser192Tyr)/p.(Arg402Gln) haplotype should be included as a pathogenic
allele in future and retrospective genetic diagnoses of OCA, supporting the idea
for a review of all previously undiagnosed OCA cases where these variants
have been excluded. Reporting of the p.(Ser192Tyr)/p.(Arg402Gln) genotype
in individuals in whom only a single deleterious TYR variant has been identified
could permit a 25-50% uplift in confirmatory molecular diagnoses (when phase
has been determined) in this diagnostically challenging patient group (Tables
3.2 and 3.3). Additionally, for patients with an albinism phenotype but no
apparent variants in albinism genes, consideration of these variants when
identified in cis as a pathogenic allele in its own right may also help provide
clinical direction. For example, in individuals heterozygous for this allele,
alternative diagnoses such as syndromic albinism might be considered less
likely, as they would be considered ‘at least a carrier of a pathogenic OCA1B
allele’, and genomic data may be re-examined in a targeted fashion to search
for further non-coding splice or structural variants in the TYR gene. Additionally,
in individuals with a very mild albinism phenotype or isolated foveal hypoplasia,
identification of this pathogenic allele in homozygous form may provide the
molecular diagnosis, ending their diagnostic odyssey.
88
Achieving an accurate molecular diagnosis will bring about important benefits
in affected individuals and their families. It allows accurate prognostic
information and family counselling to be provided, avoids the need for further
invasive investigations to confirm the clinical diagnosis or rule out syndromic
forms of the disease or masquerading conditions, and has important
therapeutic implications, given the emerging therapies currently under
development and in clinical trials for OCA (181, 182).
89
Table 3.4 Review of individuals homozygous for both TYR p.(Ser192Tyr) and p.(Arg402Gln) Individuals with an alternative molecular diagnosis responsible for the albinism phenotype were excluded from this review Study Cohort No of individuals
homozygous for
both TYR
p.(Ser192Tyr) and
p.(Arg402Gln)
Comments
Individuals with albinism with no alternative molecular diagnosis and homozygous for
p.(Ser192Tyr)/p.(Arg402Gln)
Gronskov
2019
(149)
93 individuals with a clinical
diagnosis of albinism;
diagnostic criteria included
nystagmus, reduced visual
acuity, iris translucency,
fundus hypopigmentation,
and foveal hypoplasia
6 individuals
• 5 with clinical
diagnosis of
AROA
• 1 with clinical
diagnosis of
OCA
• All 6 individuals were first
investigated by sequential Sanger
sequencing of 6 genes only (TYR,
OCA2, TYRP1, SLC45A2,
LRMDA, GPR143)
• 3 of the 6 individuals were further
investigated by whole genome
sequencing and data analysis of
TYR genomic region
Campbell
2019
(148)
12 individuals with
nystagmus, at least one
other ocular feature of
albinism, and no apparent
skin hypopigmentation in
the context of their family
2 individuals
(Proband 7 and 8;
not related)
Proband 7
• Genomic data analysis limited to
nystagmus and foveal hypoplasia
gene panel (26 genes evaluated)
• Clinical information: iris
transillumination, foveal
hypoplasia, fundal
hypopigmentation, probable VEP
crossed asymmetry
Proband 8
• Genomic data analysis limited to
ocular/oculocutaneous albinism
gene panel (18 genes evaluated)
• Rare, likely pathogenic TYRP1
variant [c.208 G>A, p.(Ala70Thr)]
also identified in this individual
• Affected brother with nystagmus,
who was homozygous for both
TYR p.(Ser192Tyr) and
p.(Arg402Gln) but WT for the
TYRP1 variant
• Clinical information: iris
transillumination, foveal
hypoplasia, fundal
hypopigmentation, VEP not
performed
This study
Salisbury cohort:
130 individuals with a
clinical diagnosis of
nystagmus and/or albinism
All were investigated with
Illumina TruSightTM One
5 individuals W1703356
• Foveal hypoplasia noted on OCT
• No other TYR or albinism-
associated variant identified
W1919237
• Afoveate adult with cystic fibrosis
and nyctalopia
90
clinical exome sequencing
followed by additional
filtering using virtual gene
panel analysis using the
“Albinism or congenital
nystagmus v1.0” Panelpp
gene panel
• No other TYR or albinism-
associated variant identified
• Biallelic ABCA4 variants also
identified and further investigation
with ERG planned; however foveal
hypoplasia is not a known feature
of ABCA4-associated retinal
dystrophy
W2002293
• Possibly afoveate (poor quality
OCT scans)
• No other TYR variant or albinism-
associated variant identified
• Maternal uncle apparently affected
with nystagmus
W1905299
• Afoveate, pale skin and hair, good
albinism phenotype
• No other TYR variant or albinism-
associated variant identified
W1817121
• Afoveate; sister also afoveate but
with no nystagmus
• No other TYR variant or albinism-
associated variant identified
Unaffected individuals homozygous for p.(Ser192Tyr)/p.(Arg402Gln)
Jagirdar
2014
(147)
2 genetic epidemiological
longitudinal studies:
Brisbane Twin Nevus
Study (1155 nuclear
families) and Queensland
Familial Melanoma Project
(1211 melanoma cases)
2 individuals Both individuals fair skinned with
fair/blond hair and blue eye colour, no
clinical diagnosis of OCA (but ocular
examination not performed)
Campbell
2019
(148)
100,000 genomes pilot
dataset of 4046 individuals
with no clinical features
suggestive of albinism
2 individuals No detailed ocular phenotyping
available
This study Southampton cohort:
161 probands with
nystagmus and/or albinism
and relatives
1 individual
This apparently unaffected individual
was father to two affected siblings who
were both clinically diagnosed with
nystagmus and/or OCA (see below).
Both affected siblings were
heterozygous for a known pathogenic
TYR variant, and also homozygous for
both TYR p.(Ser192Tyr) and
p.(Arg402Gln). This unaffected parent
was asymptomatic but was noted to
have with mild iris transillumination and
foveal hypoplasia on OCT (see Figure
3.2) in the absence of nystagmus or a
pigmentary phenotype, and had normal
visual acuities of 0.1 and 0.08 LogMAR
(right and left eye respectively).
This study Amish Exome Database:
Control exome database of
219 Amish individuals
unaffected by OCA
2 individuals No detailed ocular phenotyping
available
91
Individuals with albinism likely due to heterozygous deleterious TYR variant in combination
with homozygosity for p.(Ser192Tyr)/p.(Arg402Gln)
This study Southampton cohort:
161 probands with
nystagmus and/or albinism
and relatives
2 individuals Affected siblings, both also
heterozygous for a known pathogenic
TYR variant
This study Salisbury cohort:
130 individuals with a
clinical diagnosis of
nystagmus and/or albinism
1 individual W1809902
Also heterozygous for a TYR
pathogenic variant
Lasseaux
2018
(141)
990 index patients with at
least one of the main
characteristic ocular
features of albinism - either
nystagmus or an absence
of the fovea
1 individual TYR/R402Q-P13
Also heterozygous for a known
pathogenic TYR c.1118C>A;
p.(Thr373Lys) variant (166)
Abbreviations: AROA, autosomal recessive ocular albinism; ERG, electroretinogram; OCA, oculocutaneous albinism; OCT, optical coherence tomography; VEP, visual evoked potential; WT, wild type.
92
3.3 Genetic spectrum of OCA in Pakistan
3.3.1 Introduction
In Pakistan, geographical constraints and marriage patterns within communities
may give rise to genetic isolates in which an increased frequency of certain
disease-associated founder mutations may occur (183). Knowledge of the
specific spectrum and frequency of such genetic variants within different
regions is fundamental to the development of effective and more tailored
diagnostic genetic testing strategies, targeting variants relevant to a particular
population. This will permit rapid and cost-efficient screening and diagnostic
assays that will allow accurate disease diagnosis, improved carrier detection
and appropriate counselling for affected families.
As part of an ongoing international collaboration, genomic studies were
undertaken in a number of OCA families from several communities in Pakistan
to define the specific molecular causes of disease. In parallel with this, an
extensive literature review of previous reported genetic causes of OCA in
individuals from Pakistan was undertaken. This knowledge advances the
understanding of the relative contribution of pathogenic OCA variants in
Pakistan.
3.3.2 Materials and methods
This study entails the genetic and clinical investigations in 36 Pakistani families,
from different ethnic groups and provinces in Pakistan (Table 3.5). Medical
histories were taken from all families, and a diagnosis of OCA was established
in all affected individuals. Following results from genetic analyses and the
identification of pathogenic variants in OCA-associated disease genes, affected
members in all families were revisited, and a detailed medical history was
ascertained. Facial photographs and videos were taken with consent to
document phenotypic features and confirm disease status. Ophthalmic
examinations were completed using the best locally available resources with
support from visiting ophthalmologists from local collaborating hospitals. This
included visual acuity testing using Snellen charts and LVRC (low vision
93
resource centre) numbers – LVRC distance LogMAR Visual Acuity Chart,
colour vision testing using Ishihara charts and funduscopic examination by
direct ophthalmoscopy where possible in the affected individuals examined.
Blood samples were taken with informed consent for DNA extraction (see
section 2.3.2). As the TYR gene is a small gene comprising only five coding
exons, all families were first investigated by targeted dideoxy sequencing for
variants in one affected individual in each family, using primers designed as
described in section 2.3.2 (Appendix Table D2). This defined the likely disease-
causing variant(s) in 21 families (families 5 - 25).
In all remaining families (families 26 - 40), next generation sequencing was
performed on a single affected individual in each family using the Illumina
TruSightTM One clinical exome sequencing panel as described in section 2.3.5.
TruSightTM One clinical exome sequencing was also subsequently performed
in a single affected individual (IV:7) in family 23, and in a molecularly
undiagnosed affected individual (VI:5) in family 33. Bioinformatic analysis of
exome data was performed as per section 2.3.5. Primer design (Appendix
Table D2), PCR and dideoxy sequencing was performed as described in
section 2.3.3. to genotype and confirm appropriate segregation of the candidate
disease variant in all available affected and unaffected individuals. SNP
genotyping was performed in two affected individuals in family 40 (IV:2 and
IV:3) as described in section 2.3.4.
A literature review was performed as described in section 2.4 to retrieve all
variants and loci associated with OCA in Pakistan. Findings are summarised in
Appendix C.
3.3.3 Results: clinical and genetic findings
Study subjects from 36 families with OCA were enrolled from the Punjab, KPK
and Balochistan provinces in Pakistan. The apparent mode of inheritance in all
families was consistent with an autosomal recessive disorder. All affected
individuals displayed the cardinal clinical features of OCA with white-to-golden
94
blonde hair, pale-to-reddish white skin, decreased visual acuity, nystagmus and
photophobia. Clinical examination in all affected individuals examined
demonstrated the classical ophthalmic features of albinism, namely:
nystagmus, foveal hypoplasia and a hypopigmented albinotic fundus.
Targeted dideoxy sequencing of the TYR gene identified a number of novel and
previously reported variants in families 5 - 25 (findings summarised in Table
3.5). All identified variants appeared to segregate appropriately for autosomal
recessive disease in all families investigated (Figure 3.4), apart from family 23,
where a well reported TYR NM_000372.4:c.1037-7T>A splice variant (153,
184-190) was initially identified through targeted TYR gene sequencing in
individual IV:5 in homozygous form. This variant segregated appropriately in
affected and unaffected individuals in family 23 apart from affected individual
IV:7, in whom the TYR c.1037-7T>A variant was only detected in heterozygous
form. TruSightTM One clinical exome sequencing was subsequently performed
in this individual, which led to the identification of a further heterozygous TYR
variant, c.1255G>A; p.(Gly419Arg) that has been reported in numerous
Pakistani families residing in several regions of Pakistan including Punjab, KPK,
Sindh, and Azad Jammu and Kashmir (131, 186, 188, 191-194) (Table 3.5).
This variant was subsequently confirmed by dideoxy sequencing in individual
IV:7, but was not present in affected individuals IV:3 and IV:5.
TruSightTM One clinical exome sequencing identified a number of novel and
known variants in OCA2 in families 26 - 39, with findings summarised in Table
3.5. All identified variants segregated appropriately for an autosomal recessive
disease in all families investigated (Figure 3.4), apart from family 33, where the
previously reported OCA2 NM_000275.3:c.1045-15T>G splice variant (186,
193) was identified in homozygous form in affected individuals IV:1, IV:5, V:3,
V:8, VI:2 and VI:3, but only in heterozygous form in affected individual VI:5.
TruSightTM One clinical exome sequencing was subsequently performed in this
individual, but this did not identify any further candidate disease variants in
OCA2 or other OCA associated disease genes.
95
The novel TYR c.132T>A; p.(Ser44Arg) and c.1002delA; p.(Ala335Leufs*20)
variants were present in the gnomAD population database in heterozygous
form in 5 individuals from the Latino/admixed American population (gnomAD
v2.1.1; MAF 0.00001989) and 1 individual from the South Asian population
(gnomAD v3.1.1; MAF of 0.000006571), with no homozygous individuals
identified. The novel OCA2 c.1762C>T; p.(Arg588Trp) variant was present in
homozygous form in 5 South Asian individuals in gnomAD (v2.1.1), with a MAF
of 0.0009973 (MAF of 0.005131 in the South Asian population). The remaining
novel variants identified in this study, TYR c.240G>C; p.(Trp80Cys), TYR
c.667C>T; p.(Gln223*) and OCA2 c.2458T>C; p.(Ser820Pro) were all absent
in gnomAD (Table 3.6). In silico analysis of the novel variants identified was
undertaken using the variant prediction tools SIFT, PolyPhen-2, PROVEAN and
MutationTaster2. These were largely in agreement and predicted a deleterious
effect for all novel variants assessed, apart from OCA2 c.1762C>T;
p.(Arg588Trp), where pathogenicity predictions were conflicting (Table 3.6).
Localisation of the novel TYR and OCA2 missense variants within the
respective polypeptide is shown in Figure 3.3 alongside conservation analysis
of the affected amino acid residue across a range of vertebrate species.
In family 40, TruSightTM One clinical exome sequencing in a single affected
individual initially failed to identify any candidate disease variants likely
responsible for the OCA phenotype. Whole genome SNP mapping was
subsequently performed in both affected individuals, and identified a 7.42 Mb
region of homozygosity in chromosome 15q13.1 (GRCh38 chr15:g.22180552;
rs4462663 to chr15:g.29601735; rs1459361) common to both affected family
members and encompassing the OCA2 gene (Figure 3.5B). Array analysis
identified an intragenic homozygous deletion of ~63.4 kb within the OCA2 gene
in both affected individuals, resulting in a deletion of exon 19, and predicted to
result in a frameshift (Figure 3.5B). Exome data, analysed visually using IGV,
indicated no reads over exon 19, with a good depth of sequencing reads
covering the adjacent exons 18 and 20, again supporting a homozygous
intragenic deletion (Figure 3.5C).
96
A literature and genomic database review of known causes of OCA in families
from Pakistan was undertaken. Together with the current study, this identified
reports of 838 individuals from 197 Pakistani families with 93 candidate mapped
loci or causative genetic variants in 13 genes or loci (Appendix C), with variable
levels of confidence of causality.
97
Table 3.5 TYR and OCA2 variants segregating with albinism identified in this study
Gene Nucleotide variant
Protein variant
References gnomAD v2.1.1 MAF all/SAS (hom count)
ClinVar (Accession)
Family Province (region)
Caste
TYR c.132T>A p.(Ser44Arg) Novel 0.00001989/ Not present 5 Balochistan Khatak
NM_000372.5 0 (0) 6 KPK (Peshawar)
Pashton
c.240G>C p.(Trp80Cys) Novel Not present Not present 7 KPK Pathan
8 KPK Pathan
c.272G>A p.(Cys91Tyr) Chong et al (195) 0.000003982/ 0.00003266 (0)
Pathogenic (VCV000039977)
9 Punjab (RYK)
Somro
c.308G>A p.(Cys103Tyr) Lasseaux et al (141) Not present Not present 10 Punjab (Sahiwal)
Khokar
c.346C>T p.(Arg116*) Oettinga et al (196); Wei et al (162); Gula et al (192); Thomas et al
(144); Lin et al (197); Zhong et al (189)
0.00002829/ 0.00003266 (0)
Pathogenic (VCV000099565)
11 Punjab (Lahore)
Mughal
c.649C>T p.(Arg217Trp) Tripathi et al (198); Shahzad et al (186)
0.0001914/ 0.0003928 (0)
Pathogenic/ Likely pathogenic/ VUS (VCV000003795)
12 Punjab Rajput
c.667C>T p.(Gln223*) Novel Not present Not present 13 KPK (Bunar) Pashton
c.715C>T p.(Arg239Trp) Nakamura et al (199); Renugadevi et al (200);
Zhong et al (189)
0.00002790/ 0.00006551 (0)
Not present 14 Punjab (RYK)
Turk
c.832C>T p.(Arg278*) Tripathi et al (194); Takizawa et al (201);
Weia et al (162); Weib et al (171); Wang et al (202); Shahzad et al
(186); Lionel et al (203); Rego et al (204); Lin et
al (197); Zhong et al (189); Bibi et al (205)
0.0001699/ 0.001274 (0)
Pathogenic (VCV000099583)
15 Punjab Punjabi
16 Punjab (Lahore)
Gujjar
17 KPK (Peshawar)
Pashton
98
c.1255G>A p.(Gly419Arg) King et al (206); Chaki et al (207); Gula et al (192);
Shahzad et al (186); Gulb et al (188)
0.00006032/ 0.0003921 (0)
Pathogenic (VCV000003792)
18 Punjab (Chiniot)
Chudhar Jutt
19 Punjab (Gujranwala)
Arain
20 Punjab Virk
21 Punjab (Lahore)
Malik Awan
22 NA NA
c.1037-7T>A Affects splicing Spritza et al (184); Ribero et al (185);
Shahzad et al (186); Marti et al (153);
Ceyhan-Birsoy et al (187); Gulb et al (188); Zhong et al (189); Hou
et al (190)
0.0008614/ 0.00006551 (1)
Pathogenic/ Likely pathogenic
(VCV000099527)
23 Punjab (Sargodha)
Cheema
c.1255G>A p.(Gly419Arg) See above entry for families 18-22
See above entry for families 18-22
See above entry for families 18-22
c.832C>T p.(Arg278*) See above entry for families 15-17
See above entry for families 15-17
See above entry for families 15-17
24 Punjab (Lahore)
Mayo
c.1002delA p.(Ala335Leu fs*20)
Novel Not present Not present
c.832C>T p.(Arg278*) See above entry for families 15-17
See above entry for families 15-17
See above entry for families 15-17
25 KPK (Peshawar)
Pashton
c.1255G>A p.(Gly419Arg) See above entry for families 18-22
See above entry for families 18-22
See above entry for families 18-22
OCA2 NM_000275.3
c.1456G>T p.(Asp486Tyr) Jaworek et al (193); Shahzad et al (186)
0.00002386/ 0.0001960 (0)
VUS (VCV000617806)
26 Punjab (Qasur)
Dogar
27 Punjab (Borewala)
Dogar
28 NA NA
c.2207C>T p.(Ser736Leu) Spritzb et al (208); Marti et al (153);Yang et al
(209)
0.00002387/ 0 (0)
Likely pathogenic/ VUS
(VCV000195557)
29 NA NA
99
c.2360C>T p.(Ala787Val) Oettingb et al (210); Zhang et al (211)
0.00002475/ 0 (0)
Pathogenic/ likely pathogenic
(VCV000617810)
30 KPK (Bajaur)
NA
c.2458T>C p.(Ser820Pro) Novel Not present Not present 31 Punjab Niaz
c.1045-15T>G Affects splicing Jaworek et al (193); Shahzad et al (186)
0.00002394/ Likely pathogenic (VCV000617802)
32 Punjab Niaz
0.0001960 (0) 33 KPK Afridi
34 KPK (Peshawar)
Pashton
35 KPK (Peshawar)
Pashton
36 KPK (Peshawar)
Pashton
c.408_409delAA p.(Arg137Ilefs*83)
Novel Not present Not present 37 KPK (Peshawar)
Pashton
c.1327G>A p.(Val443Ile) Lee et al (212); Norman et al (213); Marti et al
(153); Rego et al (204); Campbell et al (214);
Hou et al (190)
0.003056/ 0.0001307 (4)
Pathogenic/ VUS (VCV000000955)
38 Punjab Saraiki
c.1762C>T p.(Arg588Trp) Novel 0.0009973/ 0.005131
(5)
VUS (VCV000885235)
c.2020C>G p.(Leu674Val) Mondal et al (215); Norman et al (213)
0.0003223/ 0.002580 (1)
Pathogenic/ VUS (VCV000194918)
39 KPK Yousafzai
c.408_409delAA p.(Arg137Ilefs*83)
See above entry for family 37
See above entry for family 37
See above entry for family 37
Exon 19 deletion Frameshift - - - 40 NA NA
Abbreviations: gnomAD, genome aggregation database; hom, homozygous; KPK, Khyber Pakhtunkhwa; MAF, minor allele frequency; RYK, Rahim Yar Khan; VUS, variant of uncertain significance. Novel variants are highlighted in red.
100
Table 3.6 Novel TYR and OCA2 variants identified in this study
Gene Variant gnomAD v2.1.1 In silico
MAF (all) MAF (SAS) Hom count SIFT PolyPhen-2 Provean MutationTaster2
TYR NM_000372.5
c.132T>A; p.(Ser44Arg)
0.00001989 0 0 Deleterious Possibly damaging
Deleterious Disease causing
c.240G>C; p.(Trp80Cys)
Not present Not present Not present Deleterious Probably damaging
Deleterious Disease causing
c.667C>T; p.(Gln223*)
Not present Not present Not present - - - Disease causing
c.1002delA; p.(Ala335Leufs*20)
Not present Not present Not present - - - Disease causing
OCA2 NM_000275.3
c.2458T>C; p.(Ser820Pro)
Not present Not present Not present Deleterious Possibly damaging
Deleterious Disease causing
c.1762C>T; p.(Arg588Trp)
0.0009973 0.005131 5 Deleterious Benign Deleterious Polymorphism
c.408_409delAA; p.(Arg137Ilefs*83)
Not present Not present Not present - - - Disease causing
Abbreviations: gnomAD, genome aggregation database; Hom, homozygous; MAF, minor allele frequency, SAS, South Asian. SIFT, Polyphen-2 and Provean in silico predictions unavailable for nonsense and frameshift variants.
101
Figure 3.3 Novel missense TYR and OCA2 variants identified in this study
Schematic showing domain architecture of A(i) TYR and B(i) OCA2 [adapted from UniProt (216)] and the location of novel missense variants identified. Abbreviations; SP, signal peptide; TM (in light blue), transmembrane domain. For the TYR polypeptide, the red diamonds denote the histidine residues that bind to copper atoms and hence structurally coordinate the positions of the copper-containing catalytic binding sites. For the OCA2 polypeptide, the citrate transporter domain is outlined in red. Conservation analysis: multiple species alignments of amino acid sequences of A(ii) TYR and B(ii) OCA2 at variant locations
102
103
104
Figure 3.4 Pedigrees and genotype data for families 5 - 39
Pedigree diagrams of families 5 - 39 investigated with OCA, showing segregation of (A) TYR and (B) OCA2 variants identified. Genotypes are shown beneath each family member investigated (+, variant; -, wild type).
105
Figure 3.5 Family 40 pedigree and OCA2 genotype data
(A) Pedigree of family 40 showing segregation of OCA2 structural variant identified. Genotypes are shown beneath each family member investigated (+, variant; -, wild type). (B) Screenshot from KaryoStudio software showing ideogram of chromosome 15 and the loss-of-heterozygosity region encompassing OCA2, shared between both affected individuals IV:2 and IV:3, as well as a homozygous deletion within this region in the OCA2 gene (C) Integrative Genome Viewer (IGV) screenshot showing absence of sequence reads over exon 19 of OCA2, with good read depth and coverage over the adjacent exons 18 and 20
3.3.4 Discussion
This study describes the findings from genetic analyses of 36 families of Pakistani
descent, identifying a total of 22 likely disease-associated variants in TYR and OCA2
responsible for OCA in Pakistani communities. These studies have identified four
novel TYR variants and three novel OCA2 variants (Table 3.6), expanding the
molecular spectrum of disease-causing OCA variants globally.
Four of the OCA-associated variants identified in this study, TYR c.1037-7T>A (family
23), and OCA2 p.(Leu674Val) (family 39), p.(Val443Ile) and p.(Arg588Trp) (both
family 38), are present in gnomAD in homozygous form in one, one, four and five
individuals respectively. The individual homozygous for TYR c.1037-7T>A is of
Ashkenazi Jewish origin, whilst the individuals in gnomAD homozygous for the above
106
OCA2 variants are all of South Asian ancestry. These variants all segregated with
disease in compound heterozygous form in their respective families. Hypomorphic
TYR variants are well defined in the aetiology of the OCA1B subtype, where reduced
tyrosinase activity results in a milder phenotype with reduced levels of pigmentation in
affected individuals. These variants may therefore represent hypomorphic variants
that cause only a partial loss of TYR or OCA2 gene function, with homozygotes
exhibiting a mild phenotype, accounting for the homozygous individuals present in the
gnomAD database.
The OCA2 p.(Leu674Val) variant has previously been described in association with
OCA in two Indian individuals in both homozygous and compound heterozygous form
(215). Both individuals exhibited an incomplete albinism phenotype, with the individual
heterozygous for the p.(Leu674Val) as well as a c.775dupG variant showing clinical
features of nystagmus, hazel irides, light golden-brown hair and pinkish skin. The
individual homozygous for the p.(Leu674Val) variant showed clinical features of brown
irides with iris transillumination, silky-brown hair and very fair pinkish skin with no
apparent nystagmus (215). This study detected the same p.(Leu674Val) variant in
compound heterozygous form in family 39, together with a novel frameshift OCA2
variant, with affected individuals again displaying an incomplete OCA phenotype with
nystagmus, blue irides, light brown hair and pink skin. The p.(Leu674Val) variant may
therefore represent a milder OCA2 mutation contributing to an incomplete OCA
phenotype when occurring in conjunction in compound heterozygous form with a more
deleterious OCA2 variant.
Although individuals homozygous for the TYR c.1037-7T>A and OCA2 p.(Val443Ile)
variants have been identified in gnomAD, these variants have also previously been
described in multiple OCA individuals in European and Chinese populations (153, 184,
189, 211, 212, 217), supporting pathogenicity. Some affected individuals compound
heterozygous for the OCA2 p.(Val443Ile) variant have been described as having a
partial albinism phenotype (213, 214), suggesting that these may be pathogenic, albeit
hypomorphic, variants. The OCA2 p.(Arg588Trp) variant affects a moderately
conserved amino acid residue, although multiple in silico prediction tools are
conflicting in their predictions of pathogenicity (Table 3.6). For affected individual II:1
in family 38 who was compound heterozygous for OCA2 p.(Arg588Trp) and
107
p.(Val443Ile) variants, re-analysis of the exome data could not identify any other
candidate variants in any of the known OCA associated genes. Ultimately, functional
characterisation of the variant would be helpful to determine the nature of the mutation
and the extent of its biological impairment (218).
In family 23, two different TYR variants [c.1037-7T>A and p.(Gly419Arg)] were
identified segregating with OCA within the same extended consanguineous family.
The presence of >1 disease-associated variant in a family may give rise to atypical
inheritance patterns or phenotypical outcomes, complicating interpretation of linkage
analysis and co-segregation results, clinical interpretation, patient counselling and
management. This intra-familial locus heterogeneity is particularly relevant within
community isolates including those within Pakistan, and has been demonstrated to
occur in up to 15% of a cohort of Pakistani families with presumed autosomal
recessive hearing loss (219). In consanguineous families with likely monogenic
diseases, where pedigree analysis suggests an autosomal recessive inheritance, but
where there is failure to identify a single rare segregating candidate disease variant in
all affected individuals, the possibility of a second disease variant or even a second
disease gene should be considered.
In family 33, the previously described disease-associated OCA2 c.1045-15T>G splice
variant was detected in six out of seven affected individuals, but only in heterozygous
form in the seventh affected individual (individual VI:5, highlighted by an (*) in Figure
3.4), with no other candidate variants in any of the known OCA associated genes
detected in this individual. Interestingly, this heterozygous OCA2 c.1045-15T>G
individual displayed a somewhat different phenotype compared to affected individuals
who were homozygous for the variant, with a darker hair colour (golden brown instead
of blonde). Unlike in family 23 described above, further genetic studies failed to identify
a second variant in the same OCA disease gene responsible for disease in this
individual. There is a high level of missing heritability in OCA, with 25% of patients
investigated only having detectable mutations in a single OCA allele (129). This may
be due to variants in the gene promoter or other regulatory regions that may not be
detected or recognised by current sequencing technologies. It is known that the
phenotype of OCA2 can be modified by MC1R or TYRP1 mutations, demonstrating a
synergistic interaction between genes throughout this pigment pathway (220, 221).
108
Possible digenic inheritance involving genetic interactions between heterozygous TYR
and OCA2 variants has also recently been described in three Pakistani OCA families
(191). Consequently, the missing heritability in this individual may reflect an
undetected mutation in an OCA disease gene or other pigment pathway gene that
interacts with OCA2. Interestingly, our research group has recently provided strong
supporting evidence for pathogenicity of a TYR haplotype consisting of a combination
of two common hypomorphic TYR variants in cis, which when inherited in trans to a
known TYR deleterious mutation, is likely to account for a proportion of OCA1B cases
with apparent missing heritability (see chapter 3.2). A similar situation may explain the
OCA2 missing heritability in family 33.
Gross deletions in the OCA2 gene are increasingly being recognised as a molecular
mechanism of disease in OCA2, with 42 such mutations reported to date (HGMD;
accessed 15.04.2021), including the 2.7 kb intragenic deletion spanning exon 7 that
is the most common cause of OCA2 in individuals of sub-Saharan African heritage
(222). Intragenic OCA2 deletions are also increasingly recognised in Pakistani
communities (140, 186, 188). Shahzad et al reported five gross deletions in OCA2,
including one family of Malick ethnicity with an exon 19 deletion (186). More recently,
a further OCA2 exon 19 deletion was reported in two further Pakistani families of
Pashtun origin recruited from Tank city, KPK, with different deletion coordinates to the
family reported by Shahzad et al (188). One limitation of this study is the inability to
accurately map breakpoints with the genomic strategies employed, and therefore it is
not possible to determine if the OCA2 exon 19 deletion in family 40 is the same as
either of those previously reported in Pakistani families. Prior studies do however
support OCA2 exon 19 deletion as a mechanism of disease, with the exon skipping
predicted to result in a frameshift and loss of function via nonsense-mediated decay.
The significant contribution of OCA2 gross deletions to OCA in Pakistani families also
supports the need for genomic or bioinformatic strategies for their detection within this
community.
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Figure 3.6 Contribution of OCA genes to OCA within Pakistan and Europe
European figures are derived from (141, 153)
The genetic studies described here, together with a comprehensive literature review
encompassing a combined cohort of 838 individuals from 197 Pakistani families
(Appendix C), highlight TYR and OCA2 as the major genes contributing to OCA in
Pakistan comparable to the prevalence in European populations (141, 153),
accounting for 50.1% and 35.4% of molecularly diagnosed cases respectively (Figure
3.6).
While most of the TYR and OCA2 variants described in this study are present in only
one or a few families, some appear to be commonly associated with OCA in Pakistan.
These include TYR variants p.(Arg278*) (identified in families 15 - 17, 24, and 25) and
p.(Gly419Arg) (identified in families 18-22, 23, 25), as well as OCA2 variants c.1045-
15T>G (identified in families 32 - 36) and p.(Asp486Tyr) (identified in families 26 - 28),
which each account for ~20% of all TYR or OCA2 alleles in Pakistani OCA families
(Appendix C). While it is not possible to conclusively determine whether common
variants represent recurrent (‘hot spot’) gene variants or regional founder variants
without more detailed genetic analyses, there is evidence in the literature to support
both mechanisms. For example, the OCA2 c.1045-15T>G and p.(Asp486Tyr) variants
have not been reported outside Pakistan, and most likely stem from a single gene
mutation event that occurred in a founder ancestor individual and which was
110
transmitted to subsequent generations, accumulating at an increased frequency in the
community. Conversely, whilst the TYR p.(Arg278*) variant appears to occur at an
increased frequency in the Pakistani population, this variant has also been identified
in numerous OCA families from various ethnicities and geographical locations,
including India (131, 215, 223-225), Guyana (194), Japan (201, 226, 227), China (162,
171, 189, 197, 202, 228-230), Korea (231), Europe (131), Mexico (131) and Israel
(232), and likely represents a recurrent gene mutation.
Founder mutations often represent important causes of disease in a particular region,
and knowledge of their presence, frequency and clinical outcomes is of enormous
value for local healthcare resource planning and for designing community-specific
diagnostic testing and counselling protocols. As with ancestral founder mutations,
knowledge of recurrent gene mutations is of great utility in the development of cost-
effective disease-specific genetic testing strategies.
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3.4 Conclusions and future work
This chapter details the significant scientific and clinical insights gained from studies
of OCA in families from Amish and Pakistani communities. The unique genomic
architecture of the Amish community, together with the relatively high frequency of
OCA type 1B in the population, enabled empowered genetic studies able to determine
the haplotype, phasing and inheritance of the hypomorphic p.(Ser192Tyr) and
p.(Arg402Gln) TYR variants in a large number of related individuals for the first time.
The pathogenicity of these two common TYR variants has been heavily debated for
some time, and this study now provides irrefutably strong evidence that the TYR
p.(Ser192Tyr) and p.(Arg402Gln) variants are pathogenic when inherited in cis. This
finding has important diagnostic implications, as considering and reporting this in-cis
haplotype as a pathogenic allele could increase the molecular diagnoses in the
diagnostically challenging albinism patient cohorts with missing heritability and/or
partial or mild albinism phenotypes. It will be crucially important to accurately
determine the phase of these common variants, and due to the high frequencies of
these variants alone in the population which can limit informative phase studies in
relatives, consideration should perhaps be given to the use of amplicon-based long-
read sequencing technologies that allow haplotype phasing in the genomic workup of
such patients (233).
Studies of families with OCA in Pakistan have identified novel TYR and OCA2 disease
variants, and expand current knowledge of the molecular spectrum and specific
genetic causes of OCA within Pakistani communities, highlighting a number of
regional founder variants. The approach adopted in this study, starting with targeted
dideoxy sequencing of TYR, followed by exome sequencing in combination with whole
genome SNP mapping studies in selected individuals, permits a rapid and cost-
effective means of achieving a molecular diagnosis in Pakistani OCA families. In
combination with existing datasets, these studies enable accurate genetic testing and
provide valuable information to aid the diagnosis and counselling of affected
individuals and family members throughout Pakistan and the wider South Asian
population.
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4 STUDIES OF CILIOPATHIES IN COMMUNITIES
4.1 Introduction
Cilia are evolutionary conserved microscopic, hair-like structures or organelles that
protrude from the cell surface. They have important and diverse biological roles in
cellular motility and the cell cycle, as well as performing extracellular sensory functions
(234). Cilia are broadly divided into two types; motile cilia, and immotile or primary
cilia. A single, non-motile (primary) cilium is present in almost every vertebrate cell,
whilst cells with motile cilia are present only in specific organs such as the reproductive
system or respiratory tract (235). The beating of motile cilia (or flagella) serves to
generate fluid movement across cell surfaces, and is responsible for the movement of
mucus up the respiratory tract, propulsion of sperm, and establishment of left-right
asymmetry in the embryonic node (234). Primary cilia on the other hand transduce
extracellular information to the intracellular environment, and have roles in
photoreception in photoreceptor cells, olfaction in olfactory neurons, and
mechanosensation of fluid flow in kidney epithelial cells (234). Primary cilia also play
an important role in several signal transduction pathways, including the noncanonical
Wnt/planar cell polarity pathway, and the Hedgehog signalling pathway (234).
The term “ciliopathy” was first used in 1984 to describe the atypical bronchial cilia
noted in a subset of children with recurrent respiratory tract infections (236). Today,
ciliopathies generally refer to a group of inherited disorders resulting from pathogenic
variants in the highly conserved genes involved in the correct assembly and
maintenance of cellular cilia or their anchoring structures, the basal bodies, resulting
in defective proteins that compromise ciliary structure or function (237, 238). To date,
pathogenic variants in approaching 200 genes have been associated with 35
ciliopathy disorders, typically segregating in an autosomal recessive form (239).
Ciliopathies arise due to dysfunction of:
• Proteins that primarily localise to - and function within - the ciliary compartment
and/or basal body (239). For example, mutations in genes encoding
components of the BBsome, an octomeric protein complex functioning as a
113
cargo adapter and utilising intraflagellar transport machinery for the trafficking
of ciliary membrane proteins, result in BBS (240).
• Proteins that are not localised within the cilia, but are required for ciliary
formation or function (239). For example, mutations in genes encoding the
cytoplasmic proteins required for pre-assembly of the multi-subunit axonemal
dynein arms in the cytoplasm prior to their transport into the cilia and flagella,
result in primary ciliary dyskinesia (PCD) (241).
Genetic defects associated with ciliopathies can affect both motile and non-motile
primary cilia, either separately or in combination (239). Additionally, some ciliary
proteins have recognised extraciliary sites and functions (242). These factors likely
contribute to the extensive phenotypic heterogeneity associated with ciliopathies
(Figure 4.1).
Ciliopathies associated primarily with impairment of motile cilia are characterised by
dysfunction of specific tissues and organs that contain motile cilia machinery. The
most common motile ciliopathy is PCD, where severe impairment of mucociliary
clearance in the respiratory epithelium leads to chronic airway disease. Additionally,
defects in ependymal cilia can result in hydrocephalus, defects in sperm flagella or in
the cilia in fallopian tubes can lead to subfertility, and cilia defects in the left-right
organiser during early embryonic development may result in laterality defects such as
situs inversus and heterotaxy (243, 244).
Defects in the sensory and/or signalling functions of primary cilia are often associated
with wide phenotypic variability compared to motile ciliopathies. These sensory
ciliopathies range from single organ disorders such as RP1 and RP1L1-associated
non-syndromic retinal dystrophy, to complex multisystem disorders such as BBS,
Joubert syndrome (JBTS) and MORM syndrome (comprising Mental retardation,
truncal Obesity, Retinal dystrophy and Micropenis) (239). This likely reflects the varied
and widespread cellular and tissue distribution of primary cilia within the human body
(245), as well as their key role in many intracellular signal transduction cascades (243).
In fact, many of the developmental anomalies associated with syndromic sensory
ciliopathies are thought to result from compromised cellular signalling, such as
polydactyly (defective Hedgehog signalling) and retinal degeneration (defective G-
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protein coupled receptor signalling) seen in BBS and JBTS (246). Retinal
degeneration is frequently observed in diverse ciliopathy syndromes (Figure 4.1),
caused by dysfunction of the retinal photoreceptor, a highly specialised neuronal cell
type whose outer segment compartment is a ciliary organelle of modified structure and
function analogous to the primary sensory cilia in other cell types (247).
Figure 4.1 Ciliopathy abacus
Schematic representation of common clinical manifestations associated with ciliopathies and their occurrence in each ciliopathy syndrome. The adding of various affected target organs (as an abacus) contributes to the specific ciliopathy syndrome. Abbreviations: BBS, Bardet-Biedl syndrome; CNS, central nervous system; JBTS, Joubert syndrome; LCA, Leber congenital amaurosis; NPHP, nephronophthisis. Image modified from (235, 248) and created with BioRender
This chapter entails a description of clinical and genomic investigations and findings
undertaken in Amish and Pakistani families with phenotypic features that are highly
suggestive of a ciliopathy disorder. While distinguishing clinical features may be
present, these (and other) phenotypically overlapping ciliopathy conditions can be
extremely difficult to differentiate clinically. This is particularly the case in developing
115
nations given the limited resources available for detailed phenotyping, and
geographical constraints restricting access to clinical services for affected families.
This work was undertaken to broaden the understanding of the molecular basis, nature
and spectrum of ciliopathy disorders in communities, and to translate that knowledge
into improved regional clinical diagnostic services for these disorders.
Within this chapter, I was responsible for the interpretation and analysis of all collected
clinical data for all affected individuals in families 41 – 44. I performed DNA extraction
for recruited individuals in family 43 (remaining DNA extraction largely completed by
Joe Leslie, University of Exeter). I was also responsible for the analysis of all exome
sequencing results, as well as primer design and subsequent cosegregation studies
for all variants identified in families within chapters 4.2 and 4.3. I performed the
literature review and analysis of all published SCAPER patients described in chapter
4.4, with a particular emphasis on delineating the ocular phenotype.
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4.2 Consolidating the phenotypic features of MORM syndrome, and
a review of INPP5E-related disorders
4.2.1 MORM syndrome
MORM syndrome is an ultra-rare ciliopathy disorder, first described in 2009 in nine
affected individuals from a single extended family in Northern Pakistan, caused by
homozygosity for a specific nonsense mutation in the INPP5E gene (249). In the same
year, homozygous missense INPP5E variants were identified in seven
consanguineous families genetically linked to the first Joubert syndrome locus
(JBTS1) on 9q34 (250). JBTS is characterised by a distinctive cerebellar and
brainstem malformation (known as the “molar tooth” sign), hypotonia in infancy with
later development of ataxia, and developmental delay, and commonly associated with
ocular involvement (including progressive retinopathy and/or coloboma) (251-253).
The cardinal clinical features of MORM syndrome include intellectual disability (mental
retardation), truncal obesity, non-progressive retinal dystrophy and micropenis (251).
Both JBTS and MORM syndrome show clinical overlap with ciliopathy syndromes,
consolidating INPP5E as a ciliopathy disease gene.
Here, clinical and genomic investigations were undertaken in an extended Pakistani
family with multiple affected individuals displaying variable phenotypic features
suggestive of a ciliopathy disorder. Molecular data and comprehensive clinical
assessments, together with a review of all previously published patients, highlight the
wide phenotypic spectrum of INPP5E-related disorders, and provide an insight into
their molecular basis.
4.2.2 Materials and methods Affected individuals underwent clinical examination at local government hospitals.
Blood samples were taken with informed consent for DNA extraction (see section
2.3.2). WES was undertaken using DNA from a single affected individual in family 41
(individual VI:3) at BGI Hong Kong, as described in section 2.3.5. Bioinformatic
analysis of exome data was performed as per section 2.3.5, with additional virtual
gene panel analysis subsequently undertaken using the “rare multisystem ciliopathy
117
disorders v1.84” PanelApp Panel (https://panelapp.genomicsengland.co.uk/).
Additional filtering was performed to retain heterozygous variants compatible with
triallelism (254). Primer design (Appendix Table D2), PCR and dideoxy sequencing
was performed as described in section 2.3.3 to genotype and confirm appropriate
segregation of the candidate disease variant in all available affected and unaffected
individuals.
A literature review was performed as described in section 2.4 to retrieve all reported
INPP5E disease-associated variants. Findings are summarised in Table 4.2.
4.2.3 Results: clinical and genetic findings
A large multigenerational extended family (family 41) with nine affected individuals
(V:1, VI:2, VI:3, VI:6, VI:7, VI:9 - 12) residing in a remote rural village in Punjab,
Pakistan, was investigated. Clinical features in affected individuals included hypotonia,
intellectual disability (mild/moderate and non-progressive), obesity, visual impairment,
aggressive and hyperactive behaviour, delayed language acquisition, speech
impairment and micropenis in males (Table 4.1).
Table 4.1 Summary of clinical features observed in affected individuals in family 41 with MORM syndrome and homozygous for the INPP5E p.(Gln627*) variant
Abbreviations: F, female; M, male; ND, no data available; yrs, years; (S), severe/profound;
(M), mild/moderate. The (✓) and (✗) symbols indicate the presence of absence of a feature in
an affected subject respectively
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After filtering WES data in a single affected individual (VI:3), only a single plausible
candidate disease variant of potential relevance to the phenotype was identified,
entailing a homozygous INPP5E nonsense variant (GRCh38) chr9:g.136429731G>A;
NM_019892.6:c.1879C>T; p.(Gln627*). This variant was absent in gnomAD (v2.1.1
and v3.1.1) and from a control exome dataset of 100 ethnically matched controls
undertaken in the Human Molecular Genetics laboratory in Pakistan. This variant is
reported as pathogenic in ClinVar (accession VCV000000396), and has previously
been reported in homozygous form in a single family with MORM syndrome from the
same province in Pakistan (249, 251). This variant segregated appropriately for an
autosomal recessive condition in the family (Figure 4.2).
Additional analysis of exome data undertaken using the “rare multisystem ciliopathy
disorders v1.84” PanelApp virtual gene panel did not identify any homozygous or
compound heterozygous candidate variants compatible with the phenotype. No
heterozygous variants compatible with triallelism were identified.
4.2.4 Discussion
INPP5E, located on chromosome 9q34.3, encodes inositol-polyphosphate 5-
phosphatase E, a 72-kDa protein with an N-terminal proline rich domain, a central 5-
phosphatase catalytic domain, and a C-terminal CaaX motif. The encoded protein
hydrolyses the 5-position phosphate from the inositol ring of the membrane‐associated
phosphatidylinositols phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] and
phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3] (255). INPP5E is widely
expressed, with enrichment in the testis and brain (255). In dividing cells, INPP5E
localises to the cytosolic face of the Golgi, possibly mediated by its N-terminal proline-
rich domain (255). In ciliated cells, the C‐terminal CaaX motif is proposed to facilitate
localisation of the protein to the primary cilium, where it is concentrated in the axoneme
(251).
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Figure 4.2 Family 41 pedigree showing INPP5E c.1879C>T genotype data
(A) Pedigree diagram of family 41 showing segregation of the INPP5E c.1879C>T; p.(Gln627*) variant, co-segregating appropriately for an autosomal recessive condition. Genotypes are shown beneath generations IV to VI (+, c.1879C>T; -, wild type). (B) Sequence chromatogram of INPP5E c.1879C>T in a homozygous affected individual. (C) Schematic showing domain architecture of INPP5E [adapted from Bielas et al (250)] and location of the c.1879C>T; p.(Gln627*) variant. IPPc; Inositol Polyphosphate Phosphatase catalytic domain. (D) Conservation analysis: multiple species alignments of the partial amino acid sequences of INPP5E in a variety of vertebrate species. The p.(Gln627*) variant is predicted to result in a truncated protein with deletion of the 18 C-terminal amino acids, including the highly conserved CaaX motif, but with preservation of the catalytic domain.
120
Inpp5e knockout mice exhibit embryonic or perinatal lethality, and exhibit features of
ciliopathy syndromes, including anophthalmia, polydactyly, polycystic kidneys,
skeletal defects and cerebral developmental defects such as anencephaly and
exencephaly (251). Post-natal Inpp5e knockout in 4-week-old mice results in further
ciliopathy features including obesity, retinal degeneration and cystic kidneys (251).
Additionally, ciliogenesis defects and abnormal cilia morphology have been noted in
Inpp5e knockout mice embryos (251) as well as in inpp5e knockdown or knockout
zebrafish embryos (256), supporting the importance of INPP5E in ciliogenesis and
cilia maintenance, and linking dysfunctional ciliary phosphoinositol metabolism to the
development of ciliopathy.
Figure 4.3 Localisation of INPP5E disease-associated variants
Schematic showing domain architecture of INPP5E protein [adapted from Bielas et al (250)] with localisation of all reported disease-associated variants to date.
Autosomal recessive pathogenic variants in INPP5E have been associated with two
different ciliopathy disorders, JBTS and MORM syndrome (Table 4.2). Pathogenic
variants in INPP5E are most commonly associated with JBTS, and these largely
missense variants are clustered adjacent to or within the 5-phosphatase catalytic
domain (Figure 4.3).
To date, there has only been a single variant associated with MORM syndrome,
[INPP5E c.1879C>T; p.(Gln627*)], reported in only a single extended Pakistani family
121
with 14 affected individuals (251) (Table 4.2). This same variant was also identified in
family 41 investigated in this study, who originate from the same province as the
original reported family, and thus likely represents a regional founder variant in the
Punjab region in Pakistan. Disease-associated INPP5E variants have also been
reported in diverse populations of different ethnicities and geographical locations
globally (Figure 4.4) (Table 4.2). In particular, two recurrent (‘hot spot’) gene variants
have been described; the INPP5E p.(Arg435Gln) variant has been reported in six
families from Turkey, Saudi Arabia, USA and Japan (250, 253, 257-259), whilst the
p.(Arg621Gln) variant has been reported in six families from Algeria, Brazil, USA,
Japan and Israel (253, 258, 260-263). Of interest, other variants disrupting the same
amino acid residue as these recurrent INPP5E variants have also been described in
affected individuals with INPP5E-associated disorders (264-266), suggesting the
arginine at amino acid positions 435 and 621 may represent clinically significant
residues.
Figure 4.4 Geographical distribution of INPP5E disease-associated variants
Size of circles relates to the number of individuals identified with the same variant in the same country. The INPP5E p.(Gln627*) MORM syndrome-associated variant is indicated by the green circle. Of note are the recurrent INPP5E p.(Arg435Gln) and p.(Arg621Gln) variants, highlighted by the red and orange circles respectively.
The affected individuals in the original MORM syndrome family reported by Hampshire
et al and Jacoby et al (249, 251) were described to have a constellation of clinical
features that were suggestive of a ciliopathy disorder, but the phenotype was thought
to be unique, and named MORM syndrome. Although patients said to have MORM
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syndrome show significant clinical overlap with other ciliopathies, the authors
distinguished MORM syndrome from JBTS and BBS on the basis of absence of
polydactyly and the nature of visual impairment in MORM syndrome patients
compared to patients with these other ciliopathy syndromes. In MORM syndrome,
visual impairment due to retinal dystrophy presents early, but is non-progressive,
whereas BBS tends to be associated with later onset retinal dystrophy and progressive
visual impairment (249). Other distinguishing features of MORM syndrome include an
absence of characteristic BBS facial features, and the presence of a small penis
without testicular anomalies (249). The clinical features observed on examination of
the nine affected individuals in this study were consistent with those in the previously
reported family, consolidating intellectual disability (mild to moderate and non-
progressive), obesity, visual impairment, aggressive and hyperactive behaviour,
hypotonia and micropenis in males as core phenotypical features of MORM syndrome
(Table 4.1).
The MORM syndrome-associated INPP5E nonsense variant is located within the last
exon of the INPP5E gene, and is therefore predicted to escape nonsense-mediated
decay, generating a prematurely truncated INPP5E protein lacking the terminal 18
amino acids with loss of the C-terminal CaaX domain (251). The CaaX motif (where C
is a cysteine reside, aa are two small generally aliphatic residues, and X is any amino
acid residue, contributing to its specificity) (267) acts as a signal for post-translational
modification by farnesylation, leading to membrane targeting and attachment of the
protein (268). Functional studies have demonstrated diminished capacity of the
MORM syndrome-associated mutant truncated protein to stabilise the primary cilium,
most likely due to a defect in ciliary localisation and protein interaction (251). Of
particular note, this mutant protein still retains its catalytic domain with no impairment
of enzymatic activity (251), in contrast to JBTS-associated missense mutations, where
the mutant protein appears to localise correctly to the cilia, but is associated with
reduced enzymatic activity (250, 269). It is tempting to postulate that this represents
the molecular basis for the genotype-phenotype correlation associated with JBTS- and
MORM-associated INPP5E mutations. Interestingly, apart from individuals with
MORM syndrome, there has only been one other affected individual identified with
biallelic truncating variants in INPP5E [homozygous for INPP5E c.1629C>G;
p.(Tyr543*)] (253). This nonsense mutation was predicted to result in the production
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of a truncated protein lacking the final part of the catalytic domain as well as the CaaX
motif (although functional studies were not performed), similar to the MORM
syndrome-associated variant, yet the affected individual had a clinical diagnosis of
JBTS with the classic “molar tooth” sign on neuroimaging pathognomic of JBTS. In
addition, De Goede et al reported an extended Pakistani family with 6 affected
individuals all homozygous for an INPP5E p.(Tyr588Cys) missense variant displaying
considerable phenotypic variability, with one individual demonstrating the classic
“molar tooth” sign on neuroimaging, suggestive of a JBTS diagnosis, whilst another
individual in the same family had a micropenis and clinical other features more
consistent with MORM syndrome (269). It is important to note that it has not been
practical to undertake neuroimaging in this study family, nor was it undertaken in the
original family reported by Hampshire et al and Jacoby et al (249, 251), and so it
remains unclear whether the cerebellum and brainstem are normal in the affected
individuals with MORM syndrome, and hence the true extent to which MORM
syndrome can be distinguished from JBTS.
Biallelic INPP5E variants have also been associated with isolated retinal dystrophies,
including Leber congenital amaurosis, retinitis pigmentosa, macular and cone-rod
dystrophy (260, 261, 266, 270-272) (Table 4.2). Interestingly, in at least four affected
individuals, systemic features were specifically excluded, suggesting that some
individuals with disease-causing INPP5E variants never develop the extraocular
manifestations typically associated with JBTS or MORM syndrome (260, 266, 270,
271, 273). None of the individuals with INPP5E-associated isolated retinal dystrophy
carry a combination of two loss-of-function variants, suggesting that complete loss of
INPP5E function will always result in a systemic phenotype (Table 4.2). Apart from
this observation, there does not seem to be a consistent correlation between an
isolated retinal dystrophy or syndromic ciliopathy phenotype, and the nature of the
variation (missense, nonsense or frameshift variants) (Figure 4.3). In fact, certain
INPP5E variants p.(Arg515Trp), p.(Arg621Gln) and p.(Tyr543*) have been associated
with both JBTS as well as isolated retinal dystrophy (250, 253, 258, 260-263, 270,
274) (Table 4.2).
Overall, these reports suggest that INPP5E variants may be associated with a wide
range of ciliopathy phenotypes. Due to the phenotypic variability associated with
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ciliopathies, it is often difficult to make diagnostic distinctions on the basis of clinical
assessment alone, particularly in developing nations due to often-limited resources
and social constraints. Identification of this INPP5E likely founder mutation will now
enable targeted genetic testing for this variant in individuals in Northern Pakistan with
an overlapping clinical presentation, permitting a rapid and cost-effective means of
achieving a molecular diagnosis.
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Table 4.2 Summary of all reported disease-associated INPP5E variants
Type of
variant
Nucleotide variant Protein variant Associated
Phenotype
Number of
reported
families
(individuals)
Country of
origin
Reference ClinVar
(Accession)
Missense c.844G>A p.(Gly282Arg) Macular
dystrophy
1 (1) Germany Birtel et al (270) VUS
(VCV000967675)
c.848C>T p.(Ala283Val) LCA 1 (1) China Wang et al (266) Not present
c.856G>A p.(Gly286Arg) JBTS 1 (1) Afghanistan Travaglini et al (253) Not present
c.874C>G p.(Arg292Gly) RP 1 (1) USA Stone et al (261) Likely pathogenic
(VCV000857108)
c.907G>A p.(Val303Met) JBTS 3 (3) Italy (2);
Uncertain (1)
Travaglini et al (253);
Toma et al (275);
Bachmann-Gagescu
et al (276)
Pathogenic/ VUS
(VCV000217653)
c.944C>T p.(Pro315Leu) JBTS 2 (2) USA (1);
Uncertain (1)
Stone et al (261);
Bachmann-Gagescu
et al (276)
Pathogenic/ VUS
(VCV000217662)
c.1021G>A p.(Gly341Ser) JBTS 2 (3) Uncertain Bachmann-Gagescu
et al (276)
Pathogenic
(VCV000217661)
c.1024T>C p.(Cys342Arg) JBTS 1 (1) China Chen et al (277) Not present
c.1035G>C p.(Arg345Ser) JBTS 1 (1) Israel Travaglini et al (253) Not present
c.1064C>T p.(Thr355Met) JBTS 2 (2) Uncertain Bachmann-Gagescu
et al (276)
Pathogenic
(VCV000217655)
c.1073C>T p.(Pro358Leu) RP 1 (1) China Xu et al (271) Not present
c.1132C>T p.(Arg378Cys) JBTS 2 (3) Italy Bielas et al (250) Pathogenic/ likely
pathogenic
(VCV000000400)
c.1133G>A p.(Arg378His) LCA 1 (1) Brazil Porto et al (260) Likely pathogenic
(VCV000866268)
c.1154G>A p.(Cys385Tyr) JBTS 1 (1) Uncertain Bachmann-Gagescu
et al (276)
Pathogenic
(VCV000217659)
126
c.1162G>T p.(Val388Leu) JBTS 1 (2) Uncertain Bachmann-Gagescu
et al (276)
Pathogenic
(VCV000217663)
c.1249T>C p.(Ser417Pro) JBTS 1 (1) Uncertain Bachmann-Gagescu
et al (276)
Pathogenic
(VCV000217664)
c.1277C>A p.(Thr426Asn) JBTS 1 (3) Switzerland Poretti et al (278) Not present
c.1303C>G p.(Arg435Gly) JBTS 1 (1) Turkey Sönmez et al (264) Not present
c.1303C>T p.(Arg435Trp) JBTS 1 (1) India Shetty et al (265) Likely pathogenic
(VCV000375472)
c.1304G>A p.(Arg435Gln) JBTS 6 (7) Turkey (2); Saudi
Arabia (1); USA
(1), Japan (1);
Uncertain (1)
Bielas et al (250);
Travaglini et al (253);
Alfares (257); Fleming
et al (258); Suzuki et
al (259); Bachmann-
Gagescu et al (276);
Pathogenic
(VCV000000399)
c.1388C>T p.(Ala463Val) JBTS 1 (1) Saudi Arabia Alfares (257) Likely pathogenic
(VCV000800892)
c.1426G>A p.(Gly476Arg) JBTS 1 (1) Japan Suzuki et al (259) Not present
c.1468G>T p.(Asp490Tyr) JBTS 1 (1) Uncertain Bachmann-Gagescu
et al (276)
Pathogenic
(VCV000217665)
c.1534C>T p.(Arg512Trp) JBTS 2 (6) Oman Bielas et al (250) Not present
c.1535G>A p.(Arg512Gln) JBTS 1 (1) Yemen Ben-Salem et al (274) VUS
(VCV000847803)
c.1543C>T p.(Arg515Trp) Cone-rod
dystrophy
1 (1) USA Stone et al (261) Pathogenic
(VCV000000397)
JBTS 3 (7) Oman Bielas et al (250);
Ben-Salem et al (274)
c.1565G>C p.(Gly522Ala) JBTS 3 (6) USA Hardee et al (252);
Fleming et al (258);
Summers et al (279)
Likely pathogenic
(VCV000530891)
c.1577C>T p.(Pro526Leu) JBTS 1 (1) Uncertain Bachmann-Gagescu
et al (276)
Pathogenic/ VUS
(VCV000217660)
c.1600T>G p.(Tyr534Asp) JBTS 1 (2) Algeria Travaglini et al (253) Not present
c.1668C>G p.(Asp556Glu) LCA 1 (1) China Wang et al (266) Not present
127
c.1669C>T p.(Arg557Cys) RP 1 (1) China Xu et al (271) Likely pathogenic
(VCV000426905)
c.1684A>G p.(Ser562Gly) JBTS 1 (2) Uncertain Bachmann-Gagescu
et al (276)
Pathogenic
(VCV000217654)
c.1688G>A p.(Arg563His) JBTS 2 (5) Turkey (1); UAE
(1)
Travaglini et al (253);
Bielas et al (250)
Pathogenic
(VCV000000398)
c.1738A>G p.(Lys580Glu) JBTS 1 (3) Egypt Bielas et al (250) Not present
c.1753C>T p.(Arg585Cys) RP 1 (1) USA Stone et al (261) Not present
JBTS 2 (3) Italy Travaglini et al (253);
Toma et al (275)
Not present
c.1754G>A p.(Arg585His) JBTS 1 (1) UAE Bachmann-Gagescu
et al (276)
Pathogenic
(VCV000217657)
c.1763A>G p.(Tyr588Cys) JBTS 1 (6) Pakistan de Goede et al (269) VUS
(VCV000635031)
c.1774C>G p.(Arg592Gly) JBTS 1 (1) USA Stone et al (261) Not present
c.1861C>T p.(Arg621Trp) LCA 1 (1) Uncertain Wang et al (273) Likely
pathogenic/ VUS
(VCV000426904)
c.1862G>A p.(Arg621Gln) LCA 2 (2) Brazil (1); Israel
(1)
Porto et al (260);
Sharon et al (263)
Likely pathogenic
(VCV000812336)
Cone-rod
dystrophy
1 (1) USA Stone et al (261)
JBTS 3 (4) Algeria (1); Japan
(1); USA (1)
Travaglini et al (253);
Tsurusaki et al (262);
Fleming et al (258)
c.1921T>C p.(Cys641Arg) JBTS 2 (4) Egypt Travaglini et al (253) Not present
Nonsense c.1629C>A p.(Tyr543*) Macular
dystrophy
1 (1) Germany Birtel et al (270) Not present
c.1629C>G p.(Tyr543*) JBTS 1 (1) Italy Travaglini et al (253) Not present
c.1879C>T p.(Gln627*) MORM
syndrome
2 (23) Pakistan Jacoby et al (251);
Khan et al (61) – this
study
Pathogenic
(VCV000000396)
128
Frameshift c.473delG p.(Gly158Valfs*40) JBTS 1 (1) USA Fleming et al (258) Likely pathogenic
(VCV000451128)
c.1760delT p.(Val587Glyfs*7) JBTS 1 (2) Uncertain Bachmann-Gagescu
et al (276)
Pathogenic
(VCV000217656)
c.1784_1787delTGAG p.(Val595Glyfs*21) JBTS 1 (1) USA Fleming et al (258) Not present
c.1897_1898delCA p.(Gln633Glufs*64) JBTS 1 (1) Uncertain Bachmann-Gagescu
et al (276)
Pathogenic
(VCV000217658)
c.700dupC p.(Leu234Profs*56) JBTS 1 (1) Japan Tsurusaki et al (262) Not present
Abbreviations: JBTS, Joubert syndrome; LCA, Leber congenital amaurosis; RP, retinitis pigmentosa; UAE; United Arab Emirates; USA, United States of America; VUS, variant of uncertain significance. Ocular disorders are highlighted in blue, and the MORM syndrome is highlighted in yellow.
129
4.2.5 A novel BBS5 variant associated with BBS in a Pakistani family
An additional multigenerational Pakistani family (family 42) residing in a remote village
in the KPK province of Pakistan was also investigated. The two affected individuals in
this family were also described as having clinical features suggestive of a ciliopathy
disorder including moderate developmental delay/intellectual disability, visual
impairment, truncal obesity, postaxial polydactyly, renal anomalies and
hypogonadism, although this was phenotypically distinct from that of family 41
described above. WES identified the likely cause of disease as a novel homozygous
frameshift mutation (NM_152384.2: c.196delA; p.(Arg66Glufs*12)) in BBS5. To date,
there have only been two BBS5 variants associated with BBS in Pakistani families,
and this finding significantly expands on the contribution of BBS5 mutations to BBS in
Pakistan. A full description of the clinical and genomic investigations performed, as
well as a comprehensive literature review of all published genetic causes of BBS in
Pakistani families, can be found in Appendix B.
130
4.3 Phenotypic heterogeneity in an extended Amish family with BBS
associated with homozygosity for the common BBS1 p.(Met390Arg)
variant
4.3.1 BBS
BBS is a rare, genetically heterogeneous, pleiotropic disorder, first described in the
early 1920s by French paediatrician Georges Louis Bardet and Hungarian pathologist
Arthur Biedl (235). BBS has an estimated prevalence of 1:125,000 - 160,000 in
Northern Europe (280, 281), but is significantly more common in certain isolated
communities such as the mixed Arab populations of Kuwait (1:36,000) (282) and in
the Newfoundland population (1:18,000) (283). Primary features of BBS include retinal
dystrophy, intellectual disability, postaxial polydactyly, obesity, renal dysfunction and
hypogonadism (284). Retinal degeneration is the most highly penetrant feature of this
condition (280, 285); in fact, after Usher syndrome, BBS is the second most common
form of syndromic retinal degeneration (261). Other variable features of BBS include
dental anomalies, cardiovascular defects, hearing loss, speech impairment, diabetes
mellitus and other limb defects (284). To date, pathogenic variants in 21 genes have
been associated with BBS in autosomal recessive or triallelic forms (286); some BBS
genes have also been implicated in other related clinical presentations including
syndromic ciliopathies as well as non-syndromic retinal dystrophies (Table 4.3).
Functionally, the majority of these genes either code for components of the core
BBSome complex (BBS1, BBS2, BBS4, BBS5, BBS7, TTC8, BBS9, BBIP1), an
octomeric protein complex involved in vesicular trafficking to the ciliary membrane
(287), or for components of the chaperonin complex (MKKS, BBS10, BBS12), which
plays an essential role in the assembly, stabilisation and regulation of the BBSome
complex (288). Other BBS-associated genes code for proteins responsible for
BBSome trafficking and localisation (ARL6, CEP290 and LZTFL1) (289-291). The
functions of some BBS genes, such as C8orf37, and their role in ciliary function, have
not yet been fully characterised (292).
131
Table 4.3 Summary of genes associated with BBS
BBS type BBS gene Allelic disorders Functions
BBS1 BBS1 - Component of BBSome
complex
BBS2 BBS2 Retinitis pigmentosa Component of BBSome
complex
BBS3 ARL6 Retinitis pigmentosa GTPase; facilitates vesicular
and interciliary trafficking
BBS4 BBS4 - Component of BBSome
complex
BBS5 BBS5 - Component of BBSome
complex
BBS6 MKKS McKusick-Kaufman syndrome Component of chaperonin
complex
BBS7 BBS7 - Component of BBSome
complex
BBS8 TTC8 Retinitis pigmentosa Component of BBSome
complex
BBS9 BBS9 - Component of BBSome
complex
BBS10 BBS10 - Component of chaperonin
complex
BBS11 TRIM32 Limb-girdle muscular dystrophy E3 Ubiquitin ligase activity
BBS12 BBS12 - Component of chaperonin
complex
BBS13 MKS1 Joubert syndrome;
Meckel syndrome
Centriole migration
BBS14 CEP290 Joubert syndrome;
Meckel syndrome;
Senior-Loken syndrome;
Leber congenital amaurosis
Interacts with RPGR
BBS15 WDPCP Congenital heart defects,
hamartomas of tongue, and
polysyndactyly syndrome
Localisation of septins and
ciliogenesis
BBS16 SDCCAG8 Senior-Loken syndrome Regulates cell polarity,
interacts with OFD1
BBS17 LZTFL1 - Negatively regulates BBSome
trafficking
BBS18 BBIP1 - Component of BBSome
complex
BBS19 IFT27 - Intraflagellar transport
BBS20 IFT74 - Ciliogenesis and length
control
BBS21 C8orf37 Retinitis pigmentosa;
Cone-rod dystrophy
Unknown
Adapted from (235, 286, 293).
132
To date, pathogenic alleles associated with overlapping ciliopathy phenotypes have
been identified in only two genes in the Amish community; the BBS1 (GRCh38)
chr11:g.66526181T>G; NM_024649.4:c.1169T>G; p.(Met390Arg) variant,
responsible for the widespread occurrence of BBS in the South-eastern Pennsylvania
Amish community (294), and the MKKS variants chr20:g.10413265G>A;
NM_170784.2:c.250C>T; p.(His84Tyr) and chr20:g.10412791C>A; c.724G>T;
p.(Ala242Ser), which segregate in cis on a haplotype that is common in the Old Order
Amish community, associated with the phenotypically distinct McKusick-Kaufman
syndrome (295).
The present study entails clinical and genomic investigations in a large Amish family
with multiple affected individuals displaying variable phenotypic features highly of
BBS, in order to aid diagnosis and clinical management.
4.3.2 Materials and methods
Affected individuals and unaffected family members from an extended Amish family
(family 43) were recruited to this study with informed consent. Blood and buccal
sample collection and DNA extraction was performed as previously described (see
section 2.3.2). WES was undertaken using DNA from a single affected individual in
the family (Individual II:1) at BGI Hong Kong, as described in section 2.3.5.
Bioinformatic analysis with additional virtual gene panel analysis and filtering to retain
heterozygous variants compatible with triallelism was performed as described in
section 4.2.2. Primer design, PCR and dideoxy sequencing (Appendix Table D2) was
also performed as previously described in section 2.3.3 to genotype and confirm
appropriate segregation of the candidate disease variant in all available affected and
unaffected individuals.
4.3.3 Results: clinical and genetic findings
A large extended Amish family (family 43) with four affected individuals (II:1, II:2, II:5
and II:10) residing in Wisconsin (USA) was investigated. Clinical features in affected
individuals included retinitis pigmentosa, polydactyly, obesity, frontal balding and
dysmorphic facies.
133
To identify the causative mutation, targeted dideoxy genotyping was first performed in
a single affected individual (II:1) for the pathogenic BBS1 and MKKS variants
previously identified in the Amish community [sequencing was only performed for the
MKKS p.(His84Tyr) variant as a proxy for the p.(His84Tyr)/(Ala242Ser) double-variant
haplotype known to be present in the Amish population] (294, 295). Genotyping results
indicated that the affected individual II:1 was homozygous for the BBS1 p.(Met390Arg)
variant and wild type for the MKKS p.(His84Tyr) variant. The BBS1 p.(Met390Arg)
variant is present in gnomAD (v2.1.1 and v3.1.1), with a MAF of 0.00157 and 0.00205
respectively, higher in the non-Finnish European population (MAF 0.002773 -
0.002895) and in the Amish population (MAF 0.0307), and also present in a single
individual in heterozygous form in a control exome dataset of 219 unrelated Amish
individuals, although there are no homozygous individuals identified in either
population database. The BBS1 p.(Met390Arg) variant alters a highly conserved
amino acid residue, and is predicted by multiple in silico tools (SIFT, Polyphen-2 and
PROVEAN) to have a deleterious effect on protein function. This variant is reported
as pathogenic or likely pathogenic in ClinVar (accession VCV000012143), and has
been reported in homozygous and compound heterozygous form in multiple
individuals with BBS (296, 297). Segregation analysis within the family confirmed that
all affected individuals were homozygous for the BBS1 p.(Met390Arg) variant;
however, homozygosity for the BBS1 p.(Met390Arg) variant was also identified in a
single apparently unaffected individual (II:7) (Figure 4.5).
WES was subsequently performed in a single affected individual (II:1) in family 43 to
identify additional variants that could be contributing to phenotypic penetrance under
the digenic triallelic inheritance model of inheritance using the “rare multisystem
ciliopathy disorders v1.84” PanelApp virtual gene panel. This confirmed homozygosity
for the BBS1 p.(Met390Arg) variant, but did not identify any further homozygous or
compound heterozygous candidate gene variants compatible with the phenotype. Two
additional candidate heterozygous variants of potential relevance were identified, a
BBS9 missense variant (GRCh38) chr7:g.33344585C>T; NM_198428.2:c.1280C>T;
p.(Ala427Val) and a CEP290 missense variant (GRCh38) chr12:g.88090784G>T;
NM_025114.3:c.3517C>A; p.(Gln1173Lys), although due to time and Covid-19
laboratory constraints, segregation studies for both were not undertaken in the wider
family.
134
Figure 4.5 Family 43 pedigree showing BBS1 p.(Met390Arg) genotype data
(A) Pedigree diagram of family 43 showing segregation of the BBS1 c.1169C>T; p.(Met390Arg) variant, co-segregating appropriately for an autosomal recessive condition (apart from the apparently unaffected individual II:7). Genotypes are shown beneath generations I and II (+, c.1169C>T; -, wild type). (B)(i) Sequence chromatogram of BBS1 c.1169C>T in a homozygous affected individual (ii) Schematic showing domain architecture of BBS1 [adapted from Chou et al (298)] and location of the c.1169T>G; p.(Met390Arg) variant. This variant is predicted to lie within the 7-bladed β-propeller domain, depicted in blue. The BBS1 protein is also predicted to contain a single α-helix as well as a 4α-helix bundle, depicted in red, and a γ-adaptin ear (GAE) domain, depicted in green. (C) Conservation analysis: multiple species alignments of partial amino acid sequences of BBS1 showing conservation of the Met390 amino acid residue across species.
The BBS9 p.(Ala427Val) variant is present in homozygous form in 6 individuals in
gnomAD (v2.1.1 and v3.1.1), with a MAF of 0.005462 - 0.005745, whilst the CEP290
p.(Gln1173Lys) variant is absent in homozygous form in both gnomAD v2.1.1 and
v3.1.1, but present in 19 and 13 individuals in heterozygous form respectively, with a
MAF of 0.00008544 - 0.00009162. Neither variant has been associated with BBS in
either autosomal recessive or triallelic forms. In silico predictions for both variants are
inconsistent with regards to variant pathogenicity. ClinVar interpretations lean towards
benign interpretation for BBS9 p.(Ala427Val) variant (accession VCV000194138),
whilst the CEP290 p.(Gln1173Lys) variant is absent in ClinVar. Overall, the clinical
significance of these two heterozygous variants remains unclear.
135
4.3.4 Discussion
BBS has traditionally been considered an autosomal recessive condition, established
by segregation analysis of large pedigrees and consanguineous families. In some
families however, more complex inheritance patterns have been proposed, which
possibly account for some of the inter- and intra-familial phenotypic variability typically
associated with BBS (299-301).
In the early 2000s, it was observed that a number of affected individuals who carried
biallelic pathogenic variants in one of the BBS genes also carried an additional
heterozygous variant in another BBS gene, and a “triallelic inheritance” model was
proposed, suggesting that in some instances a third allele was either necessary for
the disease to manifest (254, 302, 303), or instead exerted an epistatic effect on the
BBS phenotype by modifying the age of onset and/or disease severity (304, 305). A
number of BBS-associated genes have been reported to contribute to this complex
digenic triallelic inheritance pattern, including BBS1, BBS2, ARL6, BBS4, MKKS,
BBS7, BBS10, BBS12 and MKS1 (254, 303, 306-311). Additionally, variants in other
ciliary genes, including CCDC28B (312), ALMS1 (306), NPHP4 (313), TMEM67 (308),
RPGRIP1L (314), TTC21B (315) and NPHP1 (316) may also exert epistatic effects on
BBS gene mutations and modify severity of the BBS phenotype. Evidence from a
homozygous hypomorphic Cep290 mutant mouse model, where the additional loss of
Bbs4 alleles results in increased obesity and accelerated retinal degeneration
compared with mice without Bbs4 mutations, supports an interaction between
BBSome components and ciliary proteins in modifying BBS phenotypic severity (317).
An increased mutational load leading to dysfunction of more than one component in
this ciliary protein trafficking pathway could result in an additive impairment of ciliary
function, providing a plausible mechanistic basis for the variable penetrance or
phenotypic severity seen in cases of BBS with triallelic inheritance (318).
To date, however, the triallelic inheritance hypothesis in BBS remains controversial.
There are a few reports of non-penetrant individuals who carry clearly deleterious
biallelic variants in a single BBS gene, and yet are apparently unaffected (254, 303,
319, 320). Most of these reports involve the same BBS1 p.(Met390Arg) variant
136
identified in this study, and the presence of a single apparently unaffected
homozygous individual in this study (individual II:7) is therefore not unprecedented.
The BBS1 p.(Met390Arg) variant is one of the most common pathogenic variants in
the BBS1 gene, and has been reported in homozygous and compound heterozygous
state in multiple affected BBS individuals (296, 297), accounting for up to 80% of BBS1
cases in European populations (297). A Bbs1 p.(Met390Arg) knock-in mouse model
recapitulates features of the human BBS phenotype, including retinal degeneration,
male infertility and obesity (321), clearly defining this as a pathogenic BBS variant
(ClinVar accession VCV000012143.26). These findings have been used to support
the triallelic hypothesis, suggesting that at least in some cases a third pathogenic allele
is required for the disease to manifest. Indeed, Badano and coworkers initially reported
two BBS families where both the affected individual as well as the unaffected father
were found to be homozygous for the BBS1 p.(Met390Arg) variant (303). In one of
these families, the differential penetrance was subsequently explained by the epistatic
contribution of a heterozygous CCDC28B NM_024296.5:c.330C>T variant identified
in the affected individual but not in the unaffected father (312). CCDC28B codes for a
pericentriolar protein that interacts and co-localises with BBS proteins including BBS1,
BBS2, BBS4, BBS5, BBS6, BBS7, BBS8 (312). The CCDC28B c.330C>T variant is a
synonymous variant p.(Phe100Phe) that enhances the use of a cryptic splice acceptor
site, thus introducing a premature termination codon and reducing CCDC28B mRNA
levels (312). This variant however was not identified in this study.
Functional studies in zebrafish indicate the BBS1 p.(Met390Arg) variant may be a
hypomorphic variant, as evidenced by partial phenotypic rescue following introduction
of the variant into zebrafish embryos lacking BBS1 expression (322). This could
explain the milder disease phenotype associated with homozygosity for the BBS1
p.(Met390Arg) variant, with a later age of disease onset (306, 310, 323) and a clinical
presentation that may be limited to retinal degeneration with minor (polydactyly or
obesity only) or even none of the systemic features commonly associated with BBS
(319, 323-326). In fact, there have been reports of BBS1 p.(Met390Arg) homozygous
individuals in their fourth decade of life who were asymptomatic at the time of
presentation, with genetic investigations only prompted by a routine eye examination
or a relative with a retinal disease diagnosis (323). An alternative explanation for the
apparent non-penetrance of the BBS1 p.(Met390Arg) variant in individual II:7 in this
137
study (aged 12 at the time of examination) is that she may actually harbour a very mild
subclinical phenotype; a further detailed ophthalmic examination would be useful to
clarify this, although this was unfortunately not possible. It is still difficult to definitely
ascertain if there are truly non-penetrant individuals with biallelic pathogenic BBS
variants, particularly with regards to the hypomorphic BBS1 p.(Met390Arg) variant.
There are also numerous studies that do not support a triallelic inheritance pattern
modifying either penetrance or expressivity (296, 297, 327-331). Although BBS is a
rare disorder in non-endogamous populations, the large number of BBS-associated
genes suggests that the cumulative carrier frequency for at least one BBS mutation
could be as high as 1 in 50 (331), and additional rare heterozygous BBS variants in
affected individuals are possibly being detected by chance alone. Additionally, the
third alleles reported in the literature are commonly missense variants, for which
pathogenicity can sometimes be difficult to evaluate.
Adding further to the genetic complexity in BBS, there is experimental evidence that
some heterozygous modifier BBS variants may influence disease phenotype via a
dominant-negative effect, and this may even be the primary mechanism by which
modifier alleles exacerbate disease phenotype (322). This predicts that carriers of
dominant negative alleles may manifest subclinical BBS phenotypes. Consistent with
this hypothesis, BBS carriers have been reported to be at increased risk of retinal
dysfunction, obesity, hypertension, diabetes and renal disease (332-335), although
this finding has also been refuted by others (336).
Given the biological and mechanistic overlap of many BBS genes in ciliary function, it
is quite plausible that the phenotypic variability often observed in BBS could in part be
due to the modifying effects of sequence variants in genes encoding other ciliary
proteins, and in reality is likely to reflect a complex interaction between multiple genetic
factors and environmental influences. It may well be that with continued improvements
in the identification and functional validation of deep intronic, regulatory region and
copy number variants, combined with detailed phenotypic characterisations, the
impact of additional modifier gene variants in BBS can be further clarified (337, 338).
This will ultimately allow more informative counselling of affected families with regards
to prognosis and recurrence risk.
138
4.4 Delineating the expanding phenotype associated with SCAPER
gene mutation
4.4.1 SCAPER syndrome
SCAPER (S phase cyclin A–associated protein residing in the ER), encoded by the
SCAPER gene on chromosome 15q24.3, is a cyclin A/Cdk2 regulatory protein that
interacts with the cyclin A/Cdk2 complex at multiple phases of the cell cycle, and is
involved in cell cycle progression (339). A potential role for SCAPER in human disease
was first suggested by Najmabadi et al, who identified a candidate homozygous
frameshift SCAPER variant as the cause of non-syndromic intellectual disability in a
small Iranian family (340). Since then, biallelic SCAPER variants have been identified
in 24 individuals presenting with a novel syndromic disorder characterised by
intellectual disability and retinitis pigmentosa in association with variable multisystem
phenotypical presentations.
Here, clinical and genetic findings, including seven novel SCAPER variants, are
described in six individuals in five families (families 44-48) of Amish, Caucasian and
South Asian descent. Together with molecular data and comprehensive phenotypic
assessments, this enabled a more detailed clinical comparison to be drawn between
the patient cohort described [including previously published individual G001284;
Patient 3 (family 45) in this study (85)] with the 24 individuals in whom SCAPER
variants were recently defined (340-345), permitting a more precise definition of the
clinical phenotype arising from pathogenic SCAPER variation.
4.4.2 Materials and methods
Samples were taken with informed consent for DNA extraction (see section 2.3.2).
SNP genotyping was performed (Patients 1 and 2; family 44) as described in section
2.3.4. WES was undertaken as per section 2.3.5, with bioinformatics analysis, primer
design, PCR and dideoxy sequencing (Appendix Table D2) performed as described
in section 2.3.3 to genotype and confirm appropriate segregation of the candidate
disease variant in all available affected and unaffected individuals. Patient 6 (family
139
48) underwent WES at GeneDx and was identified via GeneMatcher (346) as part of
the Matchmaker Exchange Repositories (347).
A literature review was performed as described in section 2.4 to retrieve all reported
SCAPER disease-associated variants. Findings are summarised in Tables 4.4 and
4.5.
4.4.3 Results: clinical and genetic findings
Tables 4.4 and 4.5 summarise the core phenotypical features of individuals in this
study, aged between 18 months and 31 years (Patients 1 - 6; families 44 - 48), and
compares these to the clinical features of all SCAPER syndrome patients described
to date. Intellectual disability and developmental delay was present in all six affected
individuals, and four patients also exhibited hyperactivity and attention deficit
hyperactivity disorder (ADHD). Autism and dyspraxia were each noted in one
individual. Neuroimaging performed in patients 1, 3, 5 and 6 revealed no
abnormalities. Additional dysmorphic features noted in both Amish siblings (patients 1
and 2) included inverted nipples, brachydactyly, camptodactyly, proximally placed
thumbs (Figure 4.6) and a characteristic facial appearance with frontal bossing and
almond-shaped eyes; growth parameters were all normal. Patients 1, 3 - 6 all
presented between the ages of 10 - 23 with a reduction in night vision and visual field
deficits; Patient 2 described no visual symptoms at the time of presentation. Fundus
examination in patients 3 - 6 revealed findings typical of retinitis pigmentosa including
optic disc pallor, attenuated retinal vessels and intraretinal mid-peripheral bone-
spicule pigmentation and loss of photoreceptor outer segments with retained central
macular structure on OCT imaging (Figure 4.6; Table 4.5). Additional variable ocular
features described in some patients with SCAPER syndrome include cataracts (in two
individuals) and myopia and keratoconus in one individual each (Table 4.5).
140
Table 4.4 Comparison of clinical findings of all affected individuals with biallelic pathogenic SCAPER variants
Genotype Ethnicity Gender Age (yr)
Wt kg (SDS)
Ht cm (SDS)
OFC cm (SDS)
BMI (SDS)
Walked Speech delay
ID Behaviour issues
Abnormal neuro-
imaging
RP Brachy-dactyly
Other clinical findings
Najmabadi p.(Tyr118fs*)/ p.(Tyr118fs*)
Iran NA NA NA NA NA NA NA NA ✓ NA NA NA NA NA
Tatour (A:II:1)
c.2023-2A>G/ c.2023-2A>G
Arab F 24 NA NA NA NA Normal NA Mild (IQ 64)
ADHD MRI: normal ✓ NA Nil
Tatour (A:II:2)
c.2023-2A>G/ c.2023-2A>G
Arab F 23 NA NA NA NA Normal NA Mild (IQ 56)
ADHD NA ✓ NA Nil
Tatour (B:II:1)
c.2973_2976del; p.(Ile991fs*)/
c.2973_2976del; p.(Ile991fs*)
Spanish F 34 NA NA NA NA 24 mo NA Mod None reported
CT: normal ✓ NA Alopecia Areata
Tatour (C:II:4)
c.1859_1861del; p.(Glu620del)/ c.3565G>A;
p.(Ser1219Asn)
Spanish M 15 NA NA NA NA Delayed NA ✓ None reported
NA ✓ NA NA
Jauregui c.2023-2A>G/ c.2023-2A>G
Arab M 11 NA NA NA NA NA NA No No NP ✓ NA NA
Wormser (P1:V5)
c.2806del; p.(Leu936*)/ c.2806del;
p.(Leu936*)
Bedouin F 34 78 (+1.9)
145 (-3.1)
Not reduced
37.1 (+3.1)
NA ✓ Mod NA NP ✓ ✓ Genu valgum/ genu varum
Wormser (P1:V6)
c.2806del; p.(Leu936*)/ c.2806del;
p.(Leu936*)
Bedouin M 28 78 (+0.7)
157 (-3.1)
Not reduced
31.6 (+2.3)
NA ✓ Mod NA NP ✓ ✓ Genu valgum/ genu varum
Wormser (P1:V7)
c.2806del; p.(Leu936*)/ c.2806del;
p.(Leu936*)
Bedouin M 24 98 (+2.2)
163 (-2.2)
Not reduced
36.9 (+3.0)
NA ✓ Mod NA NP ✓ ✓ Genu valgum/ genu varum
Wormser (P1:V8)
c.2806del; p.(Leu936*)/ c.2806del;
p.(Leu936*)
Bedouin M 17 92 (+2.2)
155 (-2.9)
Not reduced
38.3 (+3.3)
NA ✓ Mod NA NP ✓ ✓ Genu valgum/ genu varum
Wormser (P2:III1)
c.2806del; p.(Leu936*)/ c.2806del;
p.(Leu936*)
Bedouin F 48 86.6 (+2.5)
146 (-3.0)
Not reduced
40.6 (+3.5)
NA ✓ Sev NA NP ✓ ✓ Nil
Wormser (P2:III2)
c.2806del; p.(Leu936*)/ c.2806del;
p.(Leu936*)
Bedouin F 47 62 (+0.4)
149 (-2.5)
Not reduced
27.9 (+1.6)
NA ✓ Sev NA NP ✓ ✓ Genu valgum/ genu varum
Wormser (P2:III7)
c.2806del; p.(Leu936*)/
Bedouin F 29 57.8 (+0.1)
132 (-5.2)
Not reduced
33.2 (+2.8)
NA ✓ Sev NA NP ✓ ✓ Nil
141
c.2806del; p.(Leu936*)
Wormser (P2:IV1)
c.2806del; p.(Leu936*)/ c.2806del;
p.(Leu936*)
Bedouin M 10 29.5 (-0.38)
129 (-1.5)
Not reduced
17.7 (+0.7)
NA ✓ Mod ADHD MRI: abnormal‡
suspected ✓ Genu valgum/ genu varum
Kahrizi (1:III:6)
Exon 15-16del Pakistani M 18 42 (<3rd cent)
158 (<3rd cent)
55 (13th cent)
16.8 21 mo ✓ 3 yrs
Mod (IQ 50)
Self-mutilation
MRI/ EEG: normal
✓ NA Prominent nose, narrow chin, high forehead, 2nd/3rd toe syndactyly, funnel chest, hypotonia,
polyneuropathy UL & LL, single episode febrile seizure 18 mo
Kahrizi (1:III:7)
Exon 15-16del Pakis-tani F 12 43.5 (44th cent)
150 (26th cent)
55 (76th cent)
19.1 21 mo 2 yrs Mild (IQ 67)
No MRI: mild
asymmetry lateral
ventricles
✓ NA Prominent nose, narrow chin, high
forehead, hepatomegaly
polyneuropathy UL & LL
Kahrizi # (2:II:1)
c.1096C>T; p.(Arg366*)/ c.1096C>T; p.(Arg366*)
Baloch F 34 NA 152 (-2.0)
52.5 (0)
NA 3 yrs ✓ 4 yrs
Sev (IQ 31)
No NP ✓ NA Prominent maxilla & micrognathia,
generalised tonic clonic seizures in
infancy
Kahrizi # (2:II:2)
c.1096C>T; p.(Arg366*)/ c.1096C>T; p.(Arg366*)
Baloch M 32 NA 160 (-2.3)
51 (-2.8)
NA 3 yrs ✓ 4 yrs
Sev (IQ 30)
No NP ✓ NA Prominent maxilla & micrognathia,
seizures in infancy
Kahrizi # (2:II:4)
c.1096C>T; p.(Arg366*)/ c.1096C>T; p.(Arg366*)
Baloch M 26 NA 172 (+1.0)
55 (0)
NA 3 yrs ✓ 3 yrs
Sev (IQ 34)
No NP ✓ NA Prominent maxilla,
generalised tonic clonic seizures in
infancy
Kahrizi (3:IV:1)
c.1092dup; p.(Val365fs*)/ c.1092dup;
p.(Val365fs*)
NA M 32 NA 164 (-1.8)
57.5 (+2.0)
NA 2.5 yrs 2 yrs Mod (IQ 40)
No NP ✓ NA Partial vitiligo on extremities
Kahrizi (3:IV:2)
c.1092dup; p.(Val365fs*)/ c.1092dup;
p.(Val365fs*)
NA F 12 40 145 (-1.0)
55 (+2.0)
21.4 18 mo 18 mo Mod (IQ 45)
No NP ✓ NA Nil
Kahrizi (3:IV:3)
c.1092dup; p.(Val365fs*)/ c.1092dup;
p.(Val365fs*)
NA M 20 NA 163 (0)
55 (+2.0)
NA 12 mo 24 mo Mod (IQ 40)
No NP ✓ NA Nil
142
Kahrizi (3:IV:5)
c.1092dup; p.(Val365fs*)/ c.1092dup;
p.(Val365fs*)
NA M 25 NA 164 (0)
52.5 (0)
NA 13 mo 24 mo Mod (IQ 50)
No NP ✓ NA Nil
Kahrizi (4:VI:1)
c.1883T>G; p.(Phe628Cys)/
c.1883T>G; p.(Phe628Cys)
NA F 7 15 112 (0)
49 (-2.0)
12 2.5 yrs ✓ 3 yrs
Mild (IQ 60)
No NP ✓ NA Upslanting palpebral fissures,
epicanthal fold, small mouth, low
set ears
Family 44 Patient 1
c.2236dup; p.(Ile746fs*)/ c.2236dup; p.(Ile746fs*)
Amish M 13.7 68.9 (+1.9)
166.3 (+0.7)
56.4 (+0.4)
24.9 (+2.0)
24 mo ✓ Mod Hyper-activity
MRI: normal No ✓ Proximally placed thumbs, short 5th
fingers, pes planus, frontal
bossing, almond-shaped eyes,
inverted nipples
Family 44 Patient 2
c.2236dup; p.(Ile746fs*)/ c.2236dup; p.(Ile746fs*)
Amish F 1.5 8.6 (-2.2)
78.5 (-0.7)
47.0 (-0.9)
14.0 (-2.5)
22 mo ✓ Mild Hyper-activity
NP NA (age) ✓ Proximally placed thumbs, short 5th
fingers, pes planus, frontal
bossing, almond-shaped eyes,
inverted nipples
Family 45, Patient 3*
c.2179C>T; p.(Arg727*)/ c.1116del;
p.(Val373fs*)
South Asian
F 28 25th cent
3rd cent NA NA 11 mo ✓ Mod ADHD Autism
Self-harm
MRI: norm ✓ NA Nil
Family 46, Patient 4
c.1495+1G>A/ c.3224del;
p.(Pro1075fs*)
Caucasian F 31 NA NA 57 (95th cent)
NA 15 mo ✓ Mild Dyspraxia NP ✓ NA Nil
Family 47, Patient 5
c.829C>T; p.(Arg277*)/
c.3707_3708del; p.(Ser1236fs*)
NA (USA) F 17 NA NA NA Obese NA NA ✓ NA MRI: norm ✓ NA Nil
Family 48, Patient 6
c.2377C>T; p.(Gln793*)/
c.2166-3C>G
NA (USA) F 24 63.6 (+0.6)
162.6 (-0.2)
NA 24.0 (+0.6)
15-18 ✓ Mild (IQ 50-
60)
ADHD MRI: norm ✓ NA Mod eczema with sev skin picking
behavior
Summary 8/16 obese
18/23 29/30 9/21 26/27 10/10
Abbreviations: abnorm, abnormal; ADHD, attention-deficit hyperactivity disorder; cent, centile; CT, computerised tomography; F, female; Ht, height; ID, intellectual disability; IQ, Intelligence quotient (Wechsler Adult Intelligence Scale); LL, lower limb; M, male; mo, months; Mod, moderate; MRI, magnetic resonance imaging; NA, not available; NP, not performed; OFC, occipitofrontal circumference; RP, retinitis pigmentosa; SDS, standard deviation scores; Sev, severe; susp, suspected; UL, upper limb; vent, ventricles; Wt, weight; yrs, years. (✓) indicates presence of a feature in an affected subject.
143
†Refers to age of examination ‡Abnormal MRI findings include: mildly enlarged lateral ventricles and several loci of irregular signal in the brain parenchyma above the tentorium, in the posterior white matter and along the ependyma.
*Also patient G001284 (Carss et al 2017 and Turro et al 2020 (85, 348)) #Also family M8500314 (Hu et al 2018 (344))
Height, weight, BMI and OFC Z-scores were calculated using a Microsoft Excel add-in to access growth references based on the LMS method (349) using a reference European population (350). Adults with a BMI >25 are classified as overweight, those >30 are classified as obese.
144
Table 4.5 Ocular findings of all affected individuals with biallelic pathogenic SCAPER variants
Presenting
age (yrs)
Nyctalopia visual
fields
VA logMAR
(Snellen)
Strabismus Cataract
(morphology)
Optic disc
pallor/
atrophy
Retinal
vessel
attenuation
Retinal
pigmentary
changes
CMO Electrophysiology Other ocular
findings
Najmabadi NA NA NA NA NA NA NA NA NA NA NA NA
Tatour
(A:II:1)
12-13 ✓ NA Mod/Sev NA ✓ ✓ ✓ ✓ ✓ Undetectable rod
and cone responses
Secondary
glaucoma
Tatour
(A:II:2)
12-13 ✓ NA Mod/Sev ✓ No ✓ ✓ ✓ No Undetectable rod
and cone responses
Nil
Tatour
(B:II:1)
28 ✓ ✓ Mod NA ✓
(PSC)
✓ ✓ ✓ ✓ Extinguished rod and
cone responses
Nystagmus
High myopia
Tatour
(C:II:4)
15 NA ✓ NA NA ✓ ✓ ✓ No Abolished Nil
Jauregui 9 ✓ NA RE: 0.3 (20/40)
LE: 0.1 (20/25)
NA NA ✓ ✓ ✓ No Undetectable rod,
subnormal cone
responses
Nil
Wormser
(P1:V5)
10 ✓ NA RE: PL
LE: HM
✓ ✓
(PSC)
✓ ✓ ✓ NA Extinguished rod and
cone responses
Nil
Wormser
(P1:V6)
15 ✓ NA RE: 0.3 (20/40)
LE: 0.3 (20/40)
✓ ✓
(PSC, nuclear,
punctate)
✓ ✓ ✓ NA Extinguished rod and
cone responses
Nil
Wormser
(P1:V7)
13 ✓ NA RE: 1.0 (20/200)
LE: 2.0 (20/400)
✓ ✓
(PSC)
✓ ✓ ✓ NA NA Nil
Wormser
(P1:V8)
7 ✓ NA RE: 0.24 (20/33)
LE: 0.24 (20/33)
No No NA NA Suspected NA NA Nil
Wormser
(P2:III1)
20 ✓ NA RE: NPL
LE: NPL
No ✓
(mild cortical)
✓ ✓ ✓ ✓ NA Nil
Wormser
(P2:III2)
28 ✓ NA RE: HM 15cm
LE: PL
✓ ✓
(mild PSC)
✓ ✓ ✓ ✓ NA Nil
Wormser
(P2:III7)
25 ✓ NA RE: 0.54 (20/70)
LE: 0.54 (20/70)
No No No No ✓ NA NA Nil
Wormser
(P2:IV1)
NA Unable to
assess
NA Fixes and follows
objects
No No ✓ No ✓ NA NA Nil
Kahrizi
(1:III:6)
Adolescence NA ✓ NA No No NA NA ✓ NA NA Myopia
(RE -7.25D,
LE -8.0D)
Kahrizi
(1:III:7)
12 NA NA No No NA NA ✓ NA NA Myopia
(RE -12.25D,
LE -10.25D)
145
Kahrizi #
(2:II:1)
NA NA NA No No NA NA ✓ NA NA Nil
Kahrizi #
(2:II:2)
NA NA NA NA ✓ No NA NA ✓ NA NA Nil
Kahrizi #
(2:II:4)
NA NA NA NA No No NA NA ✓ NA NA Nil
Kahrizi
(3:IV:1)
NA ✓ NA NA ✓ No NA NA ✓ NA NA Nil
Kahrizi
(3:IV:2)
NA ✓ NA NA ✓ No NA NA ✓ NA NA Nil
Kahrizi
(3:IV:3)
NA ✓ NA NA ✓ No NA NA ✓ NA NA Nil
Kahrizi
(3:IV:5)
NA ✓ NA NA ✓ No NA NA ✓ NA NA Nil
Kahrizi
(4:VI:1)
NA ✓ NA NA No NA NA ✓ NA NA Nil
Family 44
Patient 1
13 ✓ No RE: 0.48 (20/60)
LE: 0.48 (20/60)
✓ No No No No No NP Bilateral
meridonal
amblyopia
Family 44
Patient 2
NA NA NA NA NA NA NA NA NA NA NA NA
Family 45,
Patient 3*
14 ✓ ✓ RE: 0.3 (20/40)
LE: 0.3 (20/40)
NA No ✓ ✓ ✓ No Undetectable rod
responses,
pERG subnormal
Keratoconus
Family 46,
Patient 4
10 ✓ NA RE: 0.2 (20/30)
LE: 0.3 (20/40)
NA ✓ ✓ ✓ ✓ NA Undetectable rod
responses
Myopia
Family 47,
Patient 5
NA ✓ ✓ NA NA NA ✓ ✓ ✓ ✓ Severe Nil
Family 48,
Patient 6
23 Unable to
definitively
assess
✓ RE: 0.2 (20/30)
LE: 0.3 (20/40)
✓ ✓
(nuclear)
NA ✓ ✓ ✓ Undetectable rod,
severely
cone responses
Nil
Summary 20/20 5/6 12/21 9/24 14/16 14/17 26/27 6/11 11/11
Abbreviations: CMO, cystoid macular oedema; HM, detection of hand motion; LE, left eye; Mod, moderate; NA, not available; NPL; no perception of light; pERG, pattern ERG; PL, perception of light; PSC, posterior subcapsular cataract; RE, right eye; Sev, severe; VA, visual acuity; , reduced. (✓) indicates presence of a feature in an affected subject *Also patient G001284 (Carss et al 2017 and Turro et al 2020 (85, 348)). #Also family M8500314 (Hu et al 2018 (344)).
146
Patients 1 and 2 from family 44 are Ohio Amish siblings. Candidate variants identified
through WES of DNA from patient 1 were cross-referenced with regions of
autozygosity common to both affected siblings, identified through whole genome SNP
genotyping. This identified only a single plausible candidate variant, located within the
largest (18 Mb) shared region of autozygosity on chromosome 15 [rs1509805 -
rs4243078; (GRCh38) chr15:g.60281446 - 78374545], a novel homozygous
duplication variant in exon 18 of the SCAPER gene, predicted to result in a frameshift
[(GRCh38) chr15:g.76705914dupT, NM_020843.2:c.2236dupA, p.(Ile746Asnfs*6);
Figure 4.6]. Dideoxy sequencing confirmed the presence and co-segregation of this
variant in both siblings. This variant was detected in heterozygous form in five
unrelated individuals in a database of 116 regional Amish controls, corresponding to
an estimated allele frequency of ~0.04, not uncommon for founder mutations within
this population.
In patients 3 - 6, NGS identified compound heterozygous SCAPER variants
summarised in Table 4.4. The SCAPER variants in each of these patients were
confirmed to be biallelic by familial segregation analysis using dideoxy sequencing.
SCAPER variants identified in Patients 1, 2, 4 - 6 are novel. The p.(Ile746Asnfs*6) and
p.(Gln793*) SCAPER variants are present in gnomAD (v3.1.1) a single (non-Finnish)
European individual, the p.(Arg727*) variant in a single South Asian individual
(gnomAD v3.1.1), and the p.(Ser1236Tyrfs*28) in a single African/African-American
individual (gnomAD v2.1.1 and v3.1.1), all in heterozygous form only; remaining
SCAPER variants were absent from the gnomAD population database.
147
Figure 4.6 Amish (family 44) pedigree showing SCAPER c.2236dupA, p.(Ile746Asnfs*6) genotype data and selected clinical images of affected individuals
(A) Amish (family 44) pedigree (patient 1; VI:1 and patient 2; VI:5) showing segregation of the SCAPER c.2236dupA; p.(Ile746Asnfs*6) variant, co-segregating appropriately for an autosomal recessive condition. Genotypes are shown beneath generations V and VI (+, c.2236dupA; -, wild type). (B) Pictorial representation of SNP genotypes across the ~18.1 Mb chromosome 15q21-22 region identified in the Amish family. (C) Sequence chromatogram of SCAPER c.2236dupA in (i) an unaffected wild type individual and (ii) a homozygous affected individual. (D-K) Clinical features of SCAPER syndrome patients. (D-E) Brachydactyly, camptodactyly and proximally placed thumbs in Patient 1. (F-K) Ocular imaging in Patient 3 illustrating features of retinitis pigmentosa. Fundus photograph (Optos California) of right (F) and left (G) eyes showing optic disc pallor, attenuated retinal vessels and mid-peripheral bone spicule pigmentation. Fundus autofluorescence (FAF) imaging of right (H) and left (I) eyes showing mid-peripheral hypoautofluorescence with a central ring of hyperautofluorescence demarcating the surviving outer retinal structures. Optical coherence tomography (Spectralis-OCT) of right (J) and left (K) eye central retina demonstrating loss of photoreceptor outer segments with retained central macular structure corresponding to FAF findings.
4.4.4 Discussion
Here clinical and genetic studies in six affected individuals (including additional new
clinical details for Patient 3 (85)) were undertaken, which takes the total number of
SCAPER syndrome patients described to date to 30. Although the extent for which
clinical data is available for the previously reported SCAPER patients is variable, the
detailed clinical phenotyping here allows a more comprehensive clinical comparison
148
to be made with the previously reported cases, confirming the presence of a variable
pattern of dysmorphic features associated with SCAPER syndrome. It is now clear
that the cardinal clinical features of the disorder include intellectual disability and
developmental delay particularly affecting speech and language and motor
milestones. Intellectual disability in particular appears to be a remarkably penetrant
feature, and there has only been a single report of a single individual presenting with
non-syndromic retinitis pigmentosa and no evidence of intellectual disability. This
individual was found to be homozygous for a c.2023-2A>G SCAPER variant (343),
with the same variant also reported in homozygous form in two further patients with
retinitis pigmentosa, ADHD and mild intellectual disability (341). For the SCAPER
patient with apparent non-syndromic retinitis pigmentosa, collateral history from
parents and the paediatrician indicated that all developmental milestones had been
met in time with no signs or symptoms suggestive of intellectual disability, although a
formal evaluation did not appear to have been performed. There can sometimes be
difficulties in conclusively defining milder developmental delay where more subtle
clinical findings may not be identified if not specifically assessed. As such, it is still
unclear if intellectual disability is invariably present or is instead a common but variably
penetrant feature of the SCAPER syndrome.
Early adult-onset retinitis pigmentosa is another key clinical finding, and the retinal
phenotype appears remarkably consistent. In all individuals for whom data are
available, progressive loss of night vision begins in first or second decade of life.
Together with studies in mice demonstrating expression of SCAPER in multiple retinal
layers, particularly in the RPE and photoreceptor inner and outer segments, this
supports a role for SCAPER in photoreceptor function and/or maintenance (341).
Hyperactivity appears to be a common feature, with some affected individuals
receiving a formal diagnosis of ADHD. Additionally, variable forms of dysmorphic
facies are noted in eight individuals in four families, and include a prominent nose,
prominent maxilla, narrow chin or micrognathia, high forehead or frontal bossing, and
almond-shaped eyes.
Tapering fingers, brachydactyly and proximally placed thumbs, described in eight
individuals from two consanguineous Bedouin families belonging to the same tribe in
149
southern Israel, were also identified as a consistent feature in the two Amish siblings,
confirming the association of this feature with the SCAPER syndrome. Short stature
and obesity were also a common feature amongst the affected Bedouin patients, and
this constellation of clinical features including retinitis pigmentosa, obesity, short
stature, intellectual disability, developmental delay and brachydactyly has
consequently led to a suggested diagnosis of BBS in these individuals. Preliminary
functional studies have demonstrated localisation of SCAPER to primary cilia when
over-expressed in cell culture, as well as SCAPER mutations affecting length of cilia
in patient fibroblasts, suggestive of a possible role of SCAPER in ciliary dynamics and
disassembly (342). Although there is some overlap between the clinical features
characteristic of ciliopathies and those seen in SCAPER syndrome, the Amish siblings
(who are of normal height and weight for age) demonstrate that the digital, retinal and
cognitive abnormalities may occur independently of short stature and obesity. The
other common primary features of BBS, including renal anomalies, post-axial
polydactyly, hypogonadism (in males) and genital abnormalities (in females) (300),
have not been reported in association with SCAPER mutation. Whether SCAPER is
indeed a ciliary protein contributing to a ciliopathy syndrome still remains unclear.
The inverted nipples and pes planus, noted on examination of both Amish siblings,
have not been previously noted in other individuals with SCAPER variants. These
features may be unique to this family due to inherited autozygosity or may be
incompletely expressed in patients with SCAPER variants. There appears to be
significant variability in the presence and severity of extraocular features associated
with the SCAPER syndrome (Table 4.4), and detailed assessment of further patients
with respect to these features may be helpful in clarifying their possible association.
Associated ocular pathology may remain undetected or unrecognised in individuals
with intellectual disability, as such individuals often have difficulty recognising or
articulating their visual symptoms. This highlights the importance of visual screening
and ophthalmological assessment in these patients. Other common ocular features
include cataracts [in particular posterior subcapsular cataracts, which are commonly
associated with retinitis pigmentosa (351)], strabismus, and (high) myopia, with
nystagmus and keratoconus noted in a single patient. The high incidence of cataracts,
a potentially treatable cause of sight loss, as well as the association with high myopia
150
and its risk of ocular complications, again supports the case for ophthalmological
screening in early childhood.
The allele frequency (~0.04) of the Ohio Amish SCAPER c.2236dupA;
p.(Ile746Asnfs*6) founder mutation suggests that despite no previous reports, this
disorder likely represents an under-recognised cause of retinitis pigmentosa and mild
intellectual disability within this community. This highlights the importance of careful
clinical evaluation in children and adults with intellectual disability and enables
targeted genetic testing for this SCAPER variant for Amish individuals with a similar
clinical presentation, allowing a more rapid molecular diagnosis and shortening the
diagnostic odyssey for affected individuals within the community.
Together with the clinical review of all previously published patients, this study
expands the molecular spectrum of disease-causing SCAPER variants and enables a
clearer delineation of the core (and variable) phenotypical features of SCAPER
syndrome to be characterised. These findings also highlight the importance of prompt
visual screening and ophthalmic assessment in all individuals with SCAPER-
associated disease. Despite the increasing numbers of individuals identified with
SCAPER syndrome, the precise functions of SCAPER in human growth and
development are not fully understood. Further studies to elucidate the precise
molecular and developmental roles of this molecule in human growth and skeletal,
retinal and brain development and function, will yield important insights into the clinical
heterogeneity increasingly observed in SCAPER-associated disease.
151
4.5 Conclusions and future work
Four Amish and Pakistani families with overlapping clinical features suggestive of a
ciliopathy disorder underwent genetic investigations in order to provide an accurate
molecular diagnosis which could guide the clinical management of affected
individuals. These studies clearly demonstrate the utility of genomic testing in
clarifying the precise diagnosis for individuals with ciliopathy disorders.
This study reports on only the second MORM syndrome family described in literature,
and consolidates the INPP5E c.1879C>T; p.(Gln627*) likely Pakistani founder
mutation as the cause of this condition. INPP5E variants are more commonly
associated with the ciliopathy JBTS, and although MORM syndrome appears to be a
phenotypically distinct disease entity, it is still unclear if MORM syndrome should
instead be considered within the JBTS clinical spectrum. Neuroimaging studies in
MORM syndrome patients to confirm the presence or absence of the “molar tooth”
sign characteristic of JBTS would be important to clarify this. Functional studies have
described the impact of the MORM variant on INPP5E protein interaction, ciliary
localisation and catalytic activity (251). The generation of MORM and JBTS mutation-
specific murine and other animal models will be useful to further characterise any
genotype-phenotype correlation, and provide a useful resource for various cellular and
molecular studies to investigate the pathomolecular basis of disease. Given that the
MORM syndrome-associated variant is a nonsense variant, TRIDs may be a valid
therapeutic approach to explore. These drugs have already been assessed in some
ciliopathies including Usher syndrome (352) and retinitis pigmentosa (353).
Characterisation of the BBS1 p.(Met390Arg) variant in an extended Amish family
confirms the phenotypic heterogeneity associated with homozygosity for this recurrent
variant, although it did not find the evidence to either support or refute the complex
digenic triallelic inheritance hypothesis in BBS. This hypothesis should be evaluated
further, in order to provide accurate recurrence-risk estimates to at-risk family
members, and could aid in the understanding of bio-molecular pathways involved in
BBS-associated phenotypes, as well as facilitating the understanding of complex
inheritance in other genetic disorders. Zebrafish and mouse models have been widely
used in studies of ciliary dysfunction and ciliopathies including BBS (354, 355), with a
152
homozygous hypomorphic Cep290 mutant mouse model shown to demonstrate a
more severe phenotype associated with the additional loss of Bbs4 alleles (317).
Similar studies of other BBS loci in the Bbs1 Met390Arg knock-in mouse (321) and
other BBS animal models may help clarify the role of modifier genes in BBS phenotypic
expression. Further studies involving the systematic genetic screening of multiple
ciliopathy genes combined with detailed longitudinal phenotyping in large ciliopathy
patient cohorts will also be useful to define the interaction and true effects of modifier
loci, and clarify the molecular basis and disease pathways underlying the clinical
complexity in BBS and other ciliopathies. Retinal degeneration is a primary feature of
BBS, associated with a poor visual prognosis, with affected individuals often
progressing to complete blindness by 15-20 years of age (356). There have been
some early promising results from retinal gene replacement therapy studies, showing
trends towards improved retinal electrophysiological function following AAV-mediated
subretinal gene delivery in Bbs4-null and homozygous Bbs1M390R/M390R mice (357,
358). This could potentially be combined with an antiapoptotic therapy such as
systemic Tauroursodeoxycholic acid (TUDCA), which has previously been shown to
slow retinal degeneration in Bbs1M390R/M390R mice (359). Given that the majority of
BBS-associated genes code for components of the core BBSome complex or
chaperonin complex, one major challenge of gene replacement therapy however is
the need to avoid overexpression of the gene product which could disrupt protein
complex stoichiometry or lead to cell toxicity (357).
The novel SCAPER c.2236dupA; p.(Ile746Asnfs*6) founder variant identified within
the Ohio Amish population significantly expands the molecular spectrum of disease-
causing SCAPER variants, and detailed phenotyping studies in our patient cohort
enabled a clearer delineation of the core (and variable) phenotypical features of
SCAPER syndrome. Although SCAPER is now considered by some to be a BBS-
associated disease gene (301), it is still unclear whether it is indeed a ciliary protein.
Preliminary functional studies have demonstrated SCAPER localisation to primary cilia
when over-expressed in cell culture. It should be noted however that protein over-
expression in cells, particularly with a large tag that may affect protein folding, may
lead to altered localisation and may not always reflect the true endogenous location of
the protein (342). Additionally, a review of all published SCAPER syndrome patients
finds that other common primary features of ciliopathies, including obesity, renal
153
anomalies, post-axial polydactyly, hypogonadism (in males) and genital abnormalities
(in females) are not always present in affected individuals with SCAPER syndrome.
Further studies are needed to elucidate possible roles of SCAPER in primary cilia
function and dynamics. In the adult mouse retina, SCAPER localises to multiple retinal
layers, with highest expression observed in the RPE and the photoreceptor outer and
inner segments, which are terminally differentiated postmitotic cells (341). The retinal
phenotype in SCAPER syndrome does not appear to be congenital in onset, instead
presenting in the first or second decade of life. SCAPER does not therefore appear to
play a developmental role in the retina, and may instead be important for
photoreceptor function and/or maintenance. SCAPER function in the retina does not
appear to be related to its interaction with cyclin A and its putative role in cell cycle
control. Further studies to identify possible SCAPER interacting partners in the retina
may facilitate a better understanding of the underlying molecular disease pathways,
thereby allowing the development and early deployment of therapies to halt the
disease process prior to photoreceptor cell death.
154
5 IMPROVING KNOWLEDGE OF THE SPECTRUM AND
CAUSES OF RARE AND ULTRA-RARE GENETIC EYE
DISEASES IN COMMUNITIES
5.1 Introduction
Definitions of rare and ultra-rare disorders vary by country and are not well defined,
depending on factors such as disease prevalence and severity, heritability and
treatment availability (360). In Europe, rare disorders are defined as diseases or
conditions that affect fewer than 1 in 2000 people in the population, whilst ultra-rare
disorders generally refer to disorders affecting fewer than 1 in 50,000 people (or fewer
than 20 patients per million) (360, 361). It is currently estimated that there are over
6000 rare disorders (362), with new conditions continually being identified as research
advances. Although individually these disorders are uncommon, collectively they can
affect up to 30 million individuals in Europe (363) and 300 million people worldwide
(361).
Over 70% of rare diseases have an identified genetic origin (362); in fact, many genetic
disorders have a relatively low prevalence and incidence and are classified as rare or
ultra-rare disorders (364). Rare and ultra-rare disorders are often chronic conditions
associated with significant morbidity, and are also commonly difficult to diagnose and
treat. In fact, these conditions are often categorised as orphan diseases to highlight
their severity and the insufficient resources and knowledge available to develop
diagnostic assays or treatments.
There has been tremendous interest and advances in the field of rare and ultra-rare
genetic eye diseases in recent times, with increasing characterisation of causal genes
and underlying disease pathways and delineation of novel inheritance models (365).
New therapeutic approaches are entering the clinical phase of development, and the
first ever licensed gene replacement therapy for the treatment of a genetic disease in
humans, Luxturna® (voretigene neparvovec), was recently approved for the treatment
of patients with RPE65-associated retinal dystrophy (366).
155
Genetic eye diseases are however extremely difficult to study in a global setting due
to their rarity and associated phenotypic, genetic and allelic heterogeneity. The study
of inherited eye diseases in genetically isolated populations such as rural Palestinian
or Pakistani communities provides an important opportunity to learn about the genetic
causes of inherited eye diseases, whilst also providing desperately required
healthcare benefits for the families and populations involved. An improved
understanding of the underlying genetic and molecular causes of disease arising from
the identification of novel disease genes and variants within communities may then be
applied to genetically diverse populations such as the UK, and bring about clinical
benefits for patients worldwide.
In this chapter, clinical and genomic investigations were undertaken in families from
Pakistani and Palestinian communities with a preliminary clinical diagnosis of various
forms of genetic eye diseases. These studies identified 18 likely disease-causing
variants in these families, leading to an accurate molecular diagnosis and subsequent
refinement of the initial clinical diagnosis, with important treatment and prognostic
implications for affected individuals and their families.
Within this chapter, I was responsible for the interpretation and analysis of all collected
clinical data for all affected individuals (families 49 - 66). I performed DNA extraction
for a proportion of affected families (remaining DNA extraction largely completed by
Joe Leslie, University of Exeter). I was also responsible for the analysis of all exome
sequencing results, as well as primer design and subsequent cosegregation studies
for all variants identified in families within chapters 5.2 and 5.3 (apart from families 50
– 51, which performed in collaboration with Dr Shamim Saleha, Kohat University of
Science and Technology, Pakistan). I performed the literature reviews described in
this chapter (apart from review of all disease-causing ALDH1A3 variants, which was
again performed in collaboration with Dr Shamim Saleha).
156
5.2 Consolidating biallelic SDHD variants as a cause of
mitochondrial complex II deficiency
5.2.1 Introduction
The mitochondrial oxidative phosphorylation (OXPHOS) system is composed of five
multi-subunit transmembrane protein complexes (I-V) encoded by the mitochondrial
and nuclear genomes, and is the primary mechanism for adenosine triphosphate
(ATP) production in eukaryotic cells. OXPHOS defects result in mitochondrial disease,
with an estimated prevalence of 1:4300 (367, 368).
Mitochondrial complex II (succinate dehydrogenase) is composed of two catalytic
subunits (SDHA/SDHB) anchored to the inner mitochondrial membrane by two smaller
subunits (SDHC/SDHD) (369, 370). Complex II differs from other mitochondrial
respiratory chain complexes, in that its four structural subunits and their two assembly
factors (SDHAF1/SDHAF2) are solely encoded by the nuclear genome. Complex II is
also unique in being involved in both the mitochondrial respiratory chain and the Krebs
cycle (368).
Mitochondrial complex II deficiency with multisystem involvement has been reported
in association with biallelic SDHA (371), SDHB (368), SDHD (372, 373) and SDHAF1
(369, 374) gene variants, with clinical presentations including Leigh syndrome,
leukoencephalopathy, optic atrophy and cardiomyopathy with highly variable severity
and age of onset (371, 375). Complex II deficiency is rare, accounting for only 2-4%
of OXPHOS defects (372), with variants in SDHA being most common, predominantly
associated with Leigh syndrome (371). Previously, only two individuals with candidate
biallelic SDHD variants and isolated complex II deficiency have been reported (372,
373). This study describes four Palestinian siblings presenting in childhood with
clinical features indicative of mitochondrial disease and a likely pathogenic
homozygous SDHD variant, consolidating SDHD gene variants as a likely cause of
autosomal recessive mitochondrial complex II deficiency.
157
5.2.2 Materials and methods
Blood samples were collected with informed consent for DNA extraction (see section
2.3.2). SNP genotyping was performed in affected individuals V:2, V:4 and V:6 as
described in section 2.3.4. WES was undertaken using DNA from a single affected
individual (V:4) at BGI Hong Kong, as described in section 2.3.5. Primer design, PCR
and dideoxy sequencing (Appendix Table D2) was performed as described in section
2.3.3 to genotype and confirm appropriate segregation of the candidate disease
variant in all available affected and unaffected individuals.
5.2.3 Results: clinical and genetic findings
Four affected Palestinian patients (three male, one female) aged 4 - 20 years,
comprising of two sibships from an extended interconnecting family (family 49) (Figure
5.1A), were investigated in this study. All four children presented with developmental
delay in infancy and variable clinical and laboratory findings suggestive of a
mitochondrial disorder including elevated serum lactate/urinary Krebs cycle
metabolites, nystagmus, optic atrophy, progressive microcephaly, generalised
hypotonia, epileptic seizures, severe/profound intellectual disability/developmental
impairment, and cardiomyopathy. The affected children were not dysmorphic (Figure
5.1C), though individuals V:2 and V:4 were noted to have significant hypertrichosis,
particularly over their back and limbs. MRI neuroimaging was unremarkable for one
child at 8 months (V:2; Figure 5.1D), however, his sister’s scan revealed delayed
myelination at age 6 months (V:4). Hirschsprung disease, confirmed by aganglionic
rectal biopsy, was noted in a single individual (V:2). A full description of the clinical
features and disease progression are summarised in Table 5.1.
158
Figure 5.1 Family 49 pedigree showing SDHD c.205G>A genotype data, neuroimaging and images of affected individuals
(A) Pedigree diagram of family 49 showing segregation of the SDHD c.205G>A; p.(Glu69Lys) variant, co-segregating appropriately for an autosomal recessive condition. Genotypes are shown beneath generations IV and V (+, c.205G>A; -, wild type). DNA was available from all but one affected individual (V:5). (B) Electropherogram showing the DNA sequence at the position of SDHD c.205G>A in a homozygous affected individual. (C) T1 and T2-weighted axial views of MRI head of individual V:2 (aged 8 months), showing normal myelination and no intracranial abnormalities. (D) Image of affected individual V:4, illustrating the absence of any facial dysmorphism. (E)(i) Schematic showing domain architecture of SDHD [adapted from UniProt (216)] and the location of p.(Glu69Lys), p.(Asp92Gly) and p.(Ter160LeuextTer3) variants within the succinate dehydrogenase cytochrome b small subunit (CybS) domain. The orange rectangle denotes the transit peptide (TP) domain, the red circle denotes the iron (heme axial ligand) binding site shared with SDHC, and the blue circle denotes the ubiquinone binding site shared with SDHB (ii) Conservation analyses: multiple species alignments of amino acid sequences of SDHD at the locations of the p.(Glu69Lys), p.(Asp92Gly) and p.(Ter160LeuextTer3) variants. (F) Visual depiction of the two autozygous regions on chromosome 11 (shown in red) common to affected individuals V:2, V:4 and V:6 including the 2.37Mb region containing 21 genes including SDHD.
159
Table 5.1 Clinical features of affected individuals with mitochondrial complex II deficiency due to biallelic SDHD variants
Jackson et al Alston et al This study; V:2 This study; V:4 This study; V:6 This study; V:5
Genotype (NM_003002.3)
p.(Glu69Lys); (Ter160LeuextTer3)
p.(Asp92Gly); (Asp92Gly)
p.(Glu69Lys); (Glu69Lys)
p.(Glu69Lys); (Glu69Lys)
p.(Glu69Lys); (Glu69Lys)
NA (deceased)
Ethnicity Swiss Irish Palestinian Palestinian Palestinian Palestinian
Gender F M M F M M
Age at last evaluation 7 yrs (Deceased age 10 yrs)
Deceased day 1 of life from lethal
cardiomyopathy
6.4 yrs 4.5 yrs 20 yrs Hx from parents Deceased age 10 yrs -
cardiac arrest
GROWTH PARAMETERS
Birth weight kg (SDS) NA 2.62 3.5 (-0.1) 2.8 (-1.4) NA NA
Birth OFC cm (SDS) NA 34.5 35 (-0.2) 35 (+0.4) NA NA
OFC cm (SDS) 2° microcephaly from 2 yrs
NA 46.5 (-4.4) 49 (-2.0) NA NA
DEVELOPMENT
Intellectual disability Severe Maximum developmental
age of 11 mo at 4 yrs
NA Profound Profound Severe Severe
Developmental regression
✓ (from age 3 mo after
bronchiolitis) Several subsequent
episodes of regression after infection/prolonged
fasting
NA ✓ (from age 5 mo
following surgery) Previously was sitting
with support, mouthing, purposeful hand
movements
✓ (from age 4 mo)
Previously sitting with support,
fixing and following, mouthing, purposeful
hand movements
No clear hx of regression
No clear hx of regression
Gross motor skills NA NA Antigravity movements of arms and legs only
Sits with support. Rolls from back to front
Sits with support Walked with support
Fine motor skills NA NA No active hand use No active hand use Finger feed Finger fed
Expressive and receptive language development
NA NA Vocalisation, Makes sucking motions
if thirsty
Vocalisation, Responds to loud
noises
Vocalisation Points to indicate
needs
2 word phrases
Behavioural abnormalities
NA NA Sleep disturbances treated with Risperidone
None
Repetitive hand movements
-
NEUROLOGY
Generalised muscle hypotonia
✓ NA ✓ ✓ ✓ NK
160
Movement disorder Dystonia and ataxia NA NA NA Dystonia -
Seizures ✓ Polymorphic epilepsy
and intractable myoclonic movements
NA Generalised seizures post surgery
(6 mo)
Abnormal movements (4 mo)
Seizures when younger now resolved
-
EEG Normal NA Normal (7 mo) Normal NK NK
Neuroimaging Normal MRI (10 mo and 2 yrs)
NA Normal MRI (8 mo)
MRI: Delayed myelination
(6 mo)
Normal CT brain (7 yrs)
NK
OCULAR
Visual impairment ✓ NK ✓ ✓ ✓ NK
Nystagmus ✓ NK ✓ ✓ ✓ ✓
Optic atrophy ✓ NK ✓ ✓ ✓ NK
Strabismus NA NK NK ✓ ✓
HEARING IMPAIRMENT
NK
CARDIAC ABNORMALITIES
NK ✓ Hypertrophic
cardiomyopathy with Lt ventricular non-
compaction (prenatal onset)
normal cardiac
structure and function (7 yrs)
normal cardiac
structure and function (2.8 yrs)
✓ Minimal Lt ventricular hypertrophy, with low normal Lt ventricular
function (21yrs)
✓ Echo (5 yrs): Dilated cardiomyopathy with
reduced left ventricular ejection fraction, mild mitral and tricuspid
regurgitation
HYPERTRICHOSIS NK NK ✓ ✓ NK
METABOLIC INVESTIGATIONS
Raised serum lactate (10.2 mmol/L),
Lactic aciduria and ketonuria, urinary Krebs
cycle intermediates Marked defect in
complex II activity in muscle homogenate
Marked defect in complex II activity in muscle homogenate
None Raised serum lactate (5.58 mmol/L)
Urinary excretion of Krebs cycle
metabolites (succinic, fumeric and
ketoglutauric acids)
Normal respiratory chain complexes II-IV
in fibroblast homogenate
(succinate: cytochrome c reductase assay was
outside the normal range, but reported as
normal) Non-specific muscle
biopsy findings
NK
OTHER CLINICAL FEATURES
- - Hirschsprung disease diagnosed at 1 mo,
Frequent LRTI
- - -
161
Abbreviations: CT, computerised tomography; Echo, echocardiography, F, female; hx, history; M, male; mo, months; MRI, magnetic resonance imaging; NA, not available; NK, not known; OFC, occipitofrontal circumference; SDS, standard deviation scores; LRTI, lower respiratory tract infection; Lt, left; yr, years; the (✓) and () symbols indicate the presence or absence of a feature in an affected subject respectively Height, weight, BMI and OFC Z-scores were calculated using a Microsoft Excel add-in to access growth references based on the LMS method (349) using a reference European population (350)
162
Genome-wide SNP genotyping and WES was undertaken assuming that a
homozygous founder variant was responsible, although also considering other genetic
mechanisms. SNP genotyping (individuals V:2, V:4 and V:6) identified four notable (>1
Mb) shared homozygous regions, the two largest identified on chromosome 11; a
~7.00 Mb region (rs6485795 – rs11246414, chr11:g.47908294 – 54905443 [hg38]),
and a ~2.42 Mb region (rs120436 – rs12794326, chr11:g.110826521 – 113248134)
(Figure 5.1F).
WES was performed in affected individual V:4 in family 49 to identify rare functional
candidate variants. Variants were prioritised as described in section 2.3.5 and cross
referenced with SNP data, identifying only a single candidate homozygous variant of
relevance to the phenotype in SDHD [NM_003002.3:c.205G>A; p.(Glu69Lys);
chr11:g.112088902G>A], located within the second largest shared homozygous
region. This variant is present in only two heterozygotes in gnomAD (v2.1.1) and is
predicted to result in a glutamic acid to lysine substitution in an evolutionarily
conserved Glu69 residue (Figure 5.1E). This variant was previously reported as the
likely candidate cause of disease in compound heterozygous form in a single individual
with autosomal recessive encephalomyopathy and isolated mitochondrial complex II
deficiency (ClinVar accession: VCV000156153.6 and SCV001424558) (372). Dideoxy
sequencing confirmed co-segregation as appropriate for an autosomal recessive
disorder (Figure 5.1A/B).
5.2.4 Discussion
A homozygous SDHD c.205G>A; p.(Glu69Lys) missense variant was identified as the
likely cause of isolated mitochondrial complex II deficiency in three affected children
from an extended Palestinian family in this study. DNA was unavailable for individual
V:5, (deceased age 10 years), whose clinical history overlapped that of his sibling
(individual V:6). Tissues and organs heavily dependent on robust oxidative
phosphorylation processes tend to be most affected by mitochondrial disease (376),
explaining why common findings include optic atrophy, leukoencephalopathy,
myopathy, cardiomyopathy and Leigh syndrome. These clinical features overlap those
described in the affected individuals in this study, as well as the two individuals with
SDHD-related mitochondrial disease reported to date (Table 5.1).
163
Previously, compound heterozygous variants in SDHD (372) including the same
p.(Glu69Lys) variant identified here and a c.479G>T; p.(Ter160LeuextTer3) alteration
were identified as the likely candidate cause of disease in a Swiss child presenting
with developmental regression following a viral infection at three months. Progressive
ocular (visual impairment, nystagmus, optic disc pallor) and neurological (epileptic
seizures, ataxia, dystonia and continuous intractable myoclonic movement)
involvement were described, and the child died aged 10 years. Urinalysis revealed
lactic aciduria, ketonuria and Krebs cycle intermediates. Complex II activity was
deficient in skeletal muscle, and complementation studies in patient fibroblasts
showed restoration of complex II assembly and function with expression of wild-type,
but not mutant, SDHD cDNA (372). Subsequently an Irish male infant was described
(373) homozygous for a novel SDHD c.275A>G; p.(Asp92Gly) substitution, presenting
with cardiomyopathy in utero. He developed cardiopulmonary insufficiency rapidly
after birth, dying on day 1 of life. Subsequent analysis of respiratory chain function in
patient muscle homogenate revealed a marked defect in complex II activity. The four
affected individuals described here show phenotypic overlap with both these
individuals (Table 5.1).
This study extends the clinical spectrum and highlights the wide range of phenotypical
features and severity across affected individuals, even those with the same SDHD
genotype (Table 5.1). Hypertrichosis, a recognised feature of some forms of
mitochondrial disease (most notably SURF1-associated Leigh syndrome) (377), was
a noted feature in two Palestinian children. Hirschsprung disease diagnosed in a single
affected individual (V:2) has not been previously reported in association with SDHD
variants, and it remains unclear whether this is an associated or unrelated feature.
Neurodevelopmental regression is a common characteristic of mitochondrial disease,
particularly during physiologic stress through intercurrent infection, prolonged fasting
or dehydration (378). It is thus unsurprising that this appears to be a common feature
of complex II deficiency due to biallelic SDHD variants (Table 5.1). An accurate
molecular diagnosis for complex II deficient patients would support avoidance of
prolonged fasting and dehydration.
164
In addition to their role in primary mitochondrial disease, heterozygous germline
variants in SDHD and other complex II subunits and assembly factors (including
SDHA, SDHB, SDHC and SDHAF2) are associated with paragangliomas,
phaeochromocytomas and gastrointestinal stromal tumours (369). Dominantly
inherited hereditary cancer-associated SDHD variants exhibit a parent of origin effect;
typically only paternally inherited mutations are associated with disease (379, 380).
This was originally interpreted as evidence for sex-specific epigenetic modification of
the maternal SDHD allele (381); however, the SDHD gene does not appear to be
imprinted, being biallelically expressed in human brain, kidney and lymphoid tissue
(379). Instead, the ‘Hensen Model’ suggests that tumour development only occurs
following loss of function of both copies of SDHD as well as loss of an imprinted
(paternally silenced and maternally active) tumour suppressor gene likely found within
the chromosome 11p15 region, which contains several loci regulated by genomic
imprinting as well as encompassing the candidate tumour suppressor genes HK19
and CDKN1C (382, 383).
None of the three SDHD variants associated with mitochondrial complex II deficiency
have been previously linked to tumourigenesis, including in this extended Palestinian
family, although a Dutch founder familial paraganglioma SDHD variant c.274G>T;
p.(Asp92Tyr) affecting the same Asp92 amino acid residue as the p.(Asp92Gly)
mitochondrial complex II deficiency-associated variant has been described (384).
Additionally, SDHA and SDHB variants have been associated with both mitochondrial
complex II deficiency in biallelic form, and hereditary cancer susceptibility in
monoallelic form (371, 385). Therefore, routine surveillance of heterozygous SDHD
carriers is suggested for early detection of paragangliomas and phaeochromocytomas
facilitating appropriate intervention.
Together the data presented here consolidate biallelic SDHD variants as a cause of
mitochondrial disease due to mitochondrial complex II malfunction, and extend the
variable clinical features associated with the condition.
165
5.3 Informing clinical care through genomic studies in Pakistani
families with inherited ocular diseases
5.3.1 Introduction
In Pakistan, blindness presents a significant public health issue with an estimated
prevalence of 15 per 10,000 in children under the age of 15 years (386), and inherited
eye diseases are an important contributor to this burden (183, 387). Due to regional
geographical limitations or restricted local clinical resources, however, there is often
limited access to ophthalmic equipment and expertise needed for performing and
interpreting the detailed phenotyping studies needed for an accurate clinical diagnosis.
In a setting where clinical information is limited, the application of next generation
sequencing approaches may enable an accurate molecular diagnosis to be achieved,
providing important clinical benefits to affected individuals and their families, as well
as providing scientific insights into the genomic architecture of inherited eye diseases
within local communities as well as globally.
As part of an ongoing international collaboration, genomic studies were undertaken in
17 families from Pakistan with a preliminary clinical diagnosis of anophthalmia,
congenital cataracts, congenital blindness, nystagmus and OCA. This study identified
14 likely disease-causing mutations in these families, leading to an accurate molecular
diagnosis and subsequent refinement of the initial clinical diagnosis, with important
treatment and prognostic implications for affected individuals and their families.
5.3.2 Materials and methods
Genetic and clinical investigations in 17 Pakistani families were undertaken with
informed consent. Medical histories were taken to enable the initial clinical diagnosis.
Following results of genetic analyses and identification of likely pathogenic variants,
affected individuals were revisited, and a detailed medical history including
documentation of symptoms was ascertained. Clinical features were documented with
facial photographs and videos, as well as external ocular photographs. Visual acuity
testing using Snellen charts, anterior and posterior segment examinations on a slit-
166
lamp biomicroscope and B-scan ultrasonography was performed in a limited number
of affected individuals.
Blood sample collection and DNA extraction was performed as previously described
(see section 2.3.2
2.3.2 DNA extraction and quantification). WES (Exeter Sequencing Service, University of
Exeter, UK) (families 50 - 52 and 59), or Illumina TruSightTM One clinical exome
sequencing panel (families 56 - 58, 60 - 66) was performed as described in section
2.3.5 in a single affected individual in each family (apart from family 59, where two
affected individuals underwent WES). Bioinformatic analysis of exome data was
performed as per section 2.3.5. Primer design, PCR and dideoxy sequencing
(Appendix Tables D1 and D2) was performed as described in section 2.3.3 to
genotype and confirm appropriate segregation of the candidate disease variant in all
available affected and unaffected individuals.
A literature review was performed as described in section 2.4 to retrieve all reported
ALDH1A3, TDRD7 and ATOH7 inherited ocular disease-associated variants. Findings
are summarised in Tables 5.6 – 5.8.
5.3.3 Results: clinical and genetic findings
Study subjects from 17 families with individuals diagnosed with ocular conditions
alongside parents and unaffected siblings were enrolled from different provinces in
Pakistan (Sindh, KPK, Punjab and Balochistan). Two families were initially diagnosed
with anophthalmia (families 50 and 51) six families with non-syndromic congenital
cataracts (families 52 - 55, 57, 58), five families with OCA (families 62 - 66), one family
with congenital blindness (family 59) and three families with nystagmus (families 56,
60, 61). Preliminary clinical findings are summarised in Table 5.2.
Following molecular genetic findings, affected individuals underwent a further
ophthalmic examination where possible, allowing confirmation of the initial clinical
diagnosis in eight families (families 50 - 56 and family 60). In some individuals,
additional ocular pathologies were identified, allowing a refinement of the initial clinical
167
diagnoses in the remaining nine families (families 57 - 59 and 61 - 66). These results
are summarised in Table 5.2.
168
Table 5.2 Variants responsible for inherited ocular diseases identified in families 50 - 66
Family Region,
province
(Caste)
Initial clinical
diagnosis
Preliminary
ocular findings
Genotype
(affected individuals)
Previously reported gnomAD
MAF all/SAS
(hom count)
Revised
diagnosis
Additional ocular
findings post
molecular
diagnosis
50 KPK Anophthalmia Bilateral isolated
anophthalmia
ALDH1A3 c.1240G>C; p.(Gly414Arg)/ c.1240G>C; p.(Gly414Arg)
Novel Not present Isolated
anophthalmia Nil
51 KPK Anophthalmia Bilateral isolated
anophthalmia
ALDH1A3 c.172dupG; p.(Glu58Glyfs*5)/ c.172dupG; p.(Glu58Glyfs*5)
Novel Not present Isolated
anophthalmia Nil
52 Agra road,
Sindh (Jamro) Congenital
cataract Bilateral congenital
cataracts
FYCO1 c.2206C>T; p.(Gln736*)/ c.2206C>T; p.(Gln736*)
Chena et al (388); Chenb et al (389)
0.00001991/ 0.0001633
(not present)
Congenital cataract
Nil
53 Agra road,
Sindh (Jamro) Congenital
cataract Bilateral congenital
cataracts
FYCO1 c.2206C>T; p.(Gln736*)/ c.2206C>T; p.(Gln736*)
Chena et al (388); Chenb et al (389)
0.00001991/ 0.0001633
(not present)
Congenital cataract
Nil
54 Agra road,
Sindh (Jamro) Congenital
cataract Bilateral congenital
cataracts
FYCO1 c.2206C>T; p.(Gln736*)/ c.2206C>T; p.(Gln736*)
Chena et al (388); Chenb et al (389)
0.00001991/ 0.0001633
(not present)
Congenital cataract
Nil
55 Agra road,
Sindh (Jamro) Congenital
cataract Bilateral congenital
cataracts
FYCO1 c.2206C>T; p.(Gln736*)/ c.2206C>T; p.(Gln736*)
Chena et al (388); Chenb et al (389)
0.00001991/ 0.0001633
(not present)
Congenital cataract
Nil
56 KPK
(Pakhtun) Nystagmus
Nystagmus, visual deterioration,
previous intraocular surgeries
TDRD7 c.2469delG;
p.(Asn824Thrfs*27)/ c.2469delG;
p.(Asn824Thrfs*27)
Novel Not present Congenital
cataract Nil
57 Agra road,
Sindh (Jamro) Congenital
cataract Visual impairment
CYP1B1 c.1169G>A; p.(Arg390His)/ c.1169G>A; p.(Arg390His)
Stoilov et al (390); Rauf et al (391);
Sheikh et al (392)
0.0001032/ 0.0002942
(not present)
Primary congenital glaucoma
Severe optic neuropathy
58 Gambat,
Sindh (Bareejo)
Congenital cataract
Bilateral congenital cataracts
ATOH7 c.94delG; p.(Ala32Profs*55)/ c.94delG; p.(Ala32Profs*55)
Novel Not present Global ocular
developmental defects
Congenital cataract,
169
congenital glaucoma, RD
59 Attock city,
Punjab (Gujjar)
Congenital blindness
Visual impairment from birth
LRP5 c.1076C>G; p.(Thr359Arg)/ c.1076C>G; p.(Thr359Arg)
Novel Not present Global ocular
develop-mental defects
Nystagmus, microphthalmia, corneal opacity, iris adhesions,
cataract
60 KPK
(Baloch) Nystagmus
Congenital nystagmus
FRMD7 c.443T>A; p.(Leu148*)/.
Novel Not present Congenital idiopathic
nystagmus Nil
61
Quetta, Balochistan
(Pirkani, Bravi)
Nystagmus Congenital nystagmus
PAX6 c.718C>T; p.(Arg240*)/
WT
Glaser et al (6); Kondo-Saitoj et al
(393); Abouzeid et al (394); Chograni et al
(395); Primignani et al (396); Lin et al (397); Syrimis et al (398);
Cross et al (399); Tian et al (400)
Not present Isolated aniridia Aniridia
62 Balochistan
(Baloch) OCA
Nystagmus, albinism
HPS1 c.1397+1G>A/ c.1397+1G>A
Novel Not present HPS Nil
63
Shadra, Punjab (Bhatti
Rajpoot)
OCA Nystagmus,
albinism
HPS1 c.437C>T; p.(Trp146*)/ c.437C>T; p.(Trp146*)
Novel Not present HPS Nil
64
Rahim Yar Khan, Punjab
(Bukhari Sayyed)
OCA Nystagmus,
albinism
HPS1 c.972delC; p.(Met325Trpfs*6)/ c.972delC; p.(Met325Trpfs*6)
Oh et al (401) 0.0001595/
0 (not present)
HPS Nil
65 Swabi, KPK (Pakhtun, Yousafzai)
OCA Nystagmus,
albinism
HPS1 c.517C>T; p.(Arg173*)/ c.517C>T; p.(Arg173*)
Wei et al (402); Theunissen et al (403)
0.000004033/0
(not present) HPS Nil
66 Peshawar, Pakhtun
OCA Nystagmus,
albinism
HPS1 c.2009T>C; p.(Leu670Pro)/ c.2009T>C; p.(Leu670Pro)
Novel Not present HPS Nil
170
Abbreviations: gnomAD, genome aggregation database (v2.1.1); hom, homozygous; HPS, Hermansky-pudlak syndrome; KPK, Khyber Paktunkhwa; MAF, minor allele frequency; OCA, oculocutaneous albinism; SAS, South Asia; RD, retinal detachment.
Gene transcripts: ALDH1A3 (NM_000693.3); FYCO1 (NM_024513.3); TDRD7 (NM_014290.2), CYP1B1 (NM_000104.3), ATOH7 (NM_145178.3), LRP5 (NM_002335.3), FRMD7 (NM_194277.2), PAX6 (NM_000280.5), HPS1 (NM_000195.3).
171
Novel variants in ALDH1A3 associated with autosomal recessive
anophthalmia/microphthalmia
Bilateral isolated anophthalmia was the major clinical finding in all affected
individuals in families 50 and 51; affected individuals were all reported to be of
normal intelligence with no signs of intellectual disability, and no other
neurological and behavioural features were observed. WES performed in a
single affected individual in each family (family 50, individual IV:7; family 61,
individual II:1) identified homozygosity for novel ALDH1A3 variants in each
family, summarised in Table 5.2.
The novel c.1240G>C ; p.(Gly414Arg) variant identified in family 50 was absent
in gnomAD (v2.1.1 and v3.1.1); there was however another genomic variant
(c.1240G>A) resulting in the same p.(Gly414Arg) amino acid substitution that
is present in only a single heterozygous non-Finnish European individual in
gnomAD, with no homozygous individuals for this variant reported. In silico
analysis of the ALDH1A3 p.(Gly414Arg) variant using PolyPhen-2, PROVEAN
and SIFT all predict pathogenicity. In family 51, the novel c.172dupG;
p.(Glu58Glyfs*5) variant is predicted to result in a frameshift followed by a
premature stop codon [p.(Glu58Glyfs*5)], leading to loss of function via mRNA-
targeted degradation and nonsense-mediated decay. Both ALDH1A3 variants
segregate as expected for an autosomal recessive condition in each family
investigated (Figure 5.2).
A FYCO1 c.2206C>T; p.(Gln736*) likely regional founder gene variant
commonly underlies congenital cataracts in Pakistani families
In family 52, WES in single affected individual (V:7) identified a single
homozygous FYCO1 premature nonsense variant (GRCh38) c.2206C>T;
p.(Gln736*) of relevance to the ocular phenotype. This variant has previously
been described in association with congenital cataracts in Pakistani families
(388, 389). The same FYCO1 variant was subsequently identified via targeted
dideoxy sequencing in a further three congenital cataract families (families 53 -
55) residing in the same region (Agra Road, Sindh) as family 52 (Table 5.2).
172
This variant segregated as expected for an autosomal recessive condition in all
families investigated (Figure 5.3). Further clinical details are summarised in
Table 5.3.
Distinct HPS1 gene variants are associated with albinism in five Pakistani
families
Affected individuals in families 62 - 66 all had a clinical diagnosis of OCA.
TruSightTM One clinical exome sequencing of a single affected individual in
each family (family 62, individual II:2; family 63, individual IV:1; family 64,
individual IV:3; family 65, individual IV:2 and family 66, individual II:2) identified
three novel and two previous reported HPS1 variants, summarised in Table 5.2.
For the novel HPS1 c.1397+1G>A splice site variant, in silico analysis using the
bioinformatics tool Human Splicing Finder predicts an effect on splicing via
alteration of the wild-type donor site. The novel HPS1 p.(Leu670Pro) variant
alters an evolutionary conserved leucine residue (Figure 5.5), with
bioinformatics tools SIFT, PolyPhen-2 and PROVEAN all predicting
pathogenicity. Missense variants located within the same Longin 3 domain as
the p.(Leu670Pro) variant have previously been reported in association with
Hermansky-Pudlak syndrome (HPS) (141, 348, 404-408). The novel HPS1
p.(Trp146*) nonsense variant is predicted to result in loss of function via mRNA-
targeted degradation and nonsense-mediated decay.
All HPS1 variants segregated as expected for an autosomal recessive condition
in all families investigated (Figure 5.5) and were considered likely causative for
HPS in all affected individuals.
Defining sequence variants in six families with inherited ocular disease enables
refinement of clinical diagnosis
Illumina TruSightTM One clinical exome sequencing (family 56, individual II:2;
family 57, individual V:1; family 58, individual IV:1; family 60, individual IV:2;
family 61, individual IV:3) and WES (family 59, individuals IV:2 and IV:5) was
173
performed in six families with preliminary clinical diagnoses of nystagmus,
congenital cataracts and congenital blindness. In all families investigated, novel
and known likely pathogenic variants in inherited ocular disease genes that
were of plausible relevance to the ocular phenotype were identified, allowing
further refinement of the initial clinical diagnoses (summarised in Table 5.2).
The novel TDRD7 c.2469delG; p.(Asn824Thrfs*27) and ATOH7 c.94delG;
p.(Ala32Profs*55) variants are predicted to result in a frameshift. These
variants, as well as the novel FRMD7 c.443T>A, p.(Leu148*) nonsense variant,
are all predicted to result in loss of function via mRNA-targeted degradation and
nonsense-mediated decay. The novel LRP5 c.1076C>G; p.(Thr359Arg)
missense variant alters an evolutionary conserved threonine residue, with
bioinformatics tools SIFT, PolyPhen-2 and PROVEAN all predicting
pathogenicity.
Following genetic investigations, a further limited ophthalmic examination was
performed in affected individuals from families 58 and 59, with additional clinical
findings remaining consistent with the molecular diagnoses obtained (Tables
5.4 and 5.5).
In family 60, the FRMD7 p.(Leu148*) variant segregated appropriately for an X-
linked condition (penetrant in the carrier female III:2). In family 61, the PAX6
variant was found to occur de novo, and was not identified in either unaffected
parent. In the remaining families (families 57 - 59), the variants segregated
appropriately for an autosomal recessive condition. All identified variants were
thought likely to be responsible for the inherited ocular disorder in the affected
families investigated (Figure 5.4).
174
Figure 5.2 Pedigrees and ALDH1A3 genotype data for families 50 - 51
(A-B) Pedigree diagrams of families 50 and 51 showing segregation of the ALDH1A3 c.172dupG; p.(Glu58Glyfs*5) and c.1240G>C; p.(Gly414Arg) variants in family 51 (A) and family 50 (B) respectively, co-segregating appropriately for an autosomal recessive condition. Genotypes are shown beneath each family member investigated (+, variant; -, wild type). (C-D) Sequence chromatograms of a wild-type individual along with ALDH1A3 c.172dupG in a homozygous affected individual (C), as well as the DNA sequence at position ALDH1A3 c.1240G>C in a homozygous affected individual (D).
175
(E) Schematic showing domain architecture of ALDH1A3 polypeptide [predicted domains described by Moretti et al (409)] and the location of the c.172dupG; p.(Glu58Glyfs*5) and c.1240G>C; p.(Gly414Arg) variants. OD, oligomerisation domain. (F) Photographs of two affected individuals in family 49 [F(i)] and family 50 [F(i)] with non-syndromic clinical anophthalmia. (G) Conservation analysis: multiple species alignments of partial amino acid sequences of ALDH1A3 showing conservation of Glycine at position 414.
Figure 5.3 Pedigrees and FYCO1 genotype data for families 52 - 55
(A) Pedigree diagrams in families 52 - 55 showing segregation of FYCO1 c.2206C>T; p.(Gln736*) variants, co-segregating appropriately for an autosomal recessive condition. Genotypes are shown beneath each family member investigated (+, variant; -, wild type). (B) Sequence chromatograms of the FYCO1 c.2206C>T; p.(Gln736*) variant in a homozygous affected individual.
176
177
Figure 5.4 Pedigrees and genotype data for families 56 - 61
A) Pedigree diagrams of families 56 - 61 showing segregation of identified ocular disease variants. Genotypes are shown beneath each family member investigated (+, variant; -, wild type) alongside sequence chromatograms of the disease variant in a homozygous (families 56-59), hemizygous (family 60) and heterozygous (family 61) affected individual in each family. (B) Schematic showing domain architecture of LRP5 [adapted from UniProt (216)] and the location of the c.1076C>G; p.(Thr359Arg) variant. Low-density lipoprotein receptor repeat class A and B domains are denoted by the blue and green rectangles respectively, orange rectangle represents the signal peptide, red rectangles denote the coagulation Factor Xa inhibitory site, and the purple rectangle denotes the transmembrane domain. (C) Conservation analysis: multiple species alignments of partial amino acid sequences of LRP5 showing conservation of threonine at position 359.
178
Figure 5.5 Pedigrees and HPS1 genotype data for families 62 - 66
(A) Pedigree diagrams of families 62 - 66 showing segregation of identified HPS1 variants, cosegregating appropriately for an autosomal recessive condition in each family investigated. Genotypes are shown beneath each family member investigated (+, variant; -, wild type) alongside sequence chromatograms of the HPS1 variant in a homozygous affected individual in each family. (B) Schematic showing domain architecture of HPS1 [adapted from UniProt (216)] and the location of the c.2009T>C; p.(Leu670Pro) variant within the Longin 3 domain. (C) Conservation analysis: multiple species alignments of partial amino acid sequences of HPS1 showing conservation of leucine at position 670.
179
Table 5.3 Ocular findings in affected individuals homozygous for FYCO1 c.2206C>T; p.(Gln736*)
Family (ID)
Age examined
(yrs)
BCVA (Snellen)
IOP (mmHg)
Cornea AC Cataracts Cataract surgery Fundus
52 (V:3) 21 6/12 14 Clear D&Q ✓
BE present at birth
✓
IOL in situ RE, decentered LE
RE: slight optic disc pallor, healthy retina
LE: healthy optic disc & retina
52 (V:6) 13 HM 13 Clear D&Q ✓
BE present at birth
✓
IOL in situ Healthy optic disc & retina
52 (V:6) 16 PL 15 Clear D&Q ✓
BE present at birth RE lamellar cataract
✓
LE only, IOL in situ No fundal view
52 (VI:3) 3 RE: 6/18 LE: 6/9
15 Clear D&Q ✓
BE present at birth
✓
Aphakic Healthy optic disc & retina
53 (V:1) 7 RE: 6/60 LE: 6/60
RE: 11 LE: 12
Clear D&Q ✓
BE present at birth
✓
IOL in situ with PC thickening
Healthy optic disc, retina & macula
53 (V:2) 4 RE: 6/12 LE: 6/9
RE: 16 LE: 16
Clear D&Q ✓
BE present at birth
✓
IOL in situ with PC thickening
Healthy optic disc & retina
54 (V:1) 14 RE: 6/12 LE: 6/12
10 Clear D&Q ✓
BE present at birth
✓
IOL in situ with posterior capsulotomy
Healthy optic disc & retina
54 (V:3) 6 PL 8 Clear D&Q
✓
BE present at birth Lamellar and posterior
subcapsular cataract in un-operated eye
✓
Unilateral
No fundal view, dense vitritis on B-scan ultrasonography
55 (III:3) 26 RE: 6/9 LE: 6/12
9 Clear D&Q ✓
BE present at birth
✓
IOL in situ with posterior capsulotomy
Healthy optic disc & retina
55 (III:4) 23 RE: 6/12 LE: 6/9
RE: 16 LE: 16
Clear D&Q ✓
BE present at birth
✓
IOL in situ with PC thickening
Healthy optic disc & retina
180
55 (III:5) 17 RE: 6/12 LE: 6/12
13 Clear D&Q ✓
BE present at birth
✓
IOL in situ Healthy optic disc & retina
55 (IV:3) 12 PL RE: 10 LE: 10
Clear D&Q
✓
BE present at birth Cortical cataract in un-operated
eye
✓
Unilateral No fundal view
Abbreviations: AC, anterior chamber; BCVA, best corrected visual acuity; BE, both eyes; D&Q, deep and quiet; HM, hand movement; ID, individual; IOL, intraocular lens; IOP, intraocular pressure; LE, left eye; NAD, no abnormalities detected; PC, posterior capsule; PL, perception of light; RE, right eye; yrs, years.
Table 5.4 Ocular findings in affected individuals homozygous for ATOH7 c.94delG; p.(Ala32Profs*55)
Family (ID)
Age examined
(yrs)
Age of onset (yrs)
Laterality Progression BCVA (Snellen)
IOP (mmHg)
Cornea AC Lens Fundus Ocular surgery
58 (1V:1) 18 Birth Bilateral Stable HM 26 Cloudy, BK NA Cataract No view due to
cataract & BK Nil
58 (1V:3) 15 Birth Unilateral Progressing NPL Hypotony NA Shallow, phthisical
Cataract No view Glaucoma surgery
58 (1V:4) 10 Birth Unilateral Stable CF 6 Central stromal cornea opacity
Iridocorneal adhesions
IOL in situ Detached retina with multiple adhesions
Cataract surgery
58 (1V:5) 8 Birth Bilateral Stable BE: 3/60 7 BK Shallow IOL,
decentred Hypoplastic optic disc, retinal flat
Cataract surgery
58 (1V:6) 5 Birth Bilateral Progressing BE: HM 18 Cloudy,
megalocornea Shallow, PI
Clear Glaucomatous optic atrophy,
retina flat
Glaucoma surgery
Abbreviations: AC, anterior chamber; BCVA, best corrected visual acuity; BE, both eyes; BK, band keratopathy; CF, counting fingers; HM, hand movement; IOL, intraocular lens; IOP, intraocular pressure; NA, no information available; NPL, no perception of light; PI, peripheral iridotomy; PL, perception of light; yrs, years
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Table 5.5 Ocular findings in affected individuals homozygous for LRP5 c.1076C>G; p.(Thr359Arg)
Family (ID) Age examined
(yrs)
Age of onset Progression Nystagmus VA Globe Cornea Iris adhesions Cataract
59 (1V:2) 18 Early infancy Stable ✓ Blind Microphthalmia Central opacity ✓ ✓
59 (1V:5) 16 Early infancy Stable ✓ Blind Microphthalmia Central opacity ✓ ✓
59 (1V:7) 15 Early infancy Stable ✓ Blind Microphthalmia Central opacity ✓ ✓
59 (1V:9) 17 Early infancy Stable ✓ Blind Microphthalmia Central opacity ✓ ✓
59 (IV:13) 15 Early infancy Stable ✓ Blind Microphthalmia Central opacity ✓ ✓
Abbreviations: VA, visual acuity; yrs, years. (✓) denotes the presence of a feature
182
5.3.4 Discussion
Anophthalmia and microphthalmia are severe congenital developmental defects of the
eye. In the clinical context, anophthalmia refers to a complete absence of the globe in
the orbit, whilst microphthalmia refers to the presence of a small globe within the orbit.
Both anophthalmia and microphthalmia are more commonly bilateral, although they
can also present unilaterally. These are relatively rare defects, occurring with an
estimated combined incidence of 1 in 10,000 live births (410). Anophthalmia and
microphthalmia can occur as isolated malformations, or as part of a syndrome; both
forms have been associated with autosomal recessive, autosomal dominant and X-
linked patterns of inheritance, and display extensive genetic heterogeneity (411).
SOX2 variants are the major single-gene cause of anophthalmia and microphthalmia,
accounting for ~10 - 15% of all cases (412). Variants in other genes have been shown
to account for another ~25% of cases of anophthalmia and microphthalmia (413). In
up to 50 - 60% of cases however, the underlying genetic cause remains undetermined
(411, 414).
Fares-Taie et al first provided evidence in 2013 for ALDH1A3 involvement in
autosomal recessive anophthalmia and microphthalmia in humans (412). Since then,
including the individuals reported in this study, ALDH1A3 variants have been identified
as a cause of autosomal recessive anophthalmia and microphthalmia in 67 individuals
from 28 families to date (Table 5.6). Among these, 56 individuals are from 19
consanguineous families (412, 415-425) and even where consanguinity was not
documented, most affected individuals were found to be homozygous for the disease-
causing ALDH1A3 variant, with only two reported individuals with anophthalma or
microphthalmia associated with compound heterozygosity for pathogenic ALDH1A3
variants (426, 427).
ALDH1A3-associated anophthalmia and microphthalmia is frequently reported in
association with other ocular and extraocular anomalies, such as the presence of short
eyelids, blepharophimosis and reduced palpebral fissures (420, 421, 425, 426),
entropion (425), conjunctival symblepharon (421), conjunctival discoloration (421),
large eyebrows and synophrys (421, 423), coloboma (416, 417, 419, 421, 425),
hypoplasia of the optic tracts and chiasm (412, 415, 421, 423, 425), hypoplastic extra
183
ocular muscles (415, 423), refractive errors including both myopia and hyperopia (416,
417), and esotropia (416). There is a high variability observed in the phenotypic
expression of dysmorphic or systemic features associated with anophthalmia and
microphthalmia, even in individuals with the same ALDH1A3 gene variants (418, 423,
424). Mild hypoplasia of the vermis (variant of Dandy-Walker malformation), as well
as pulmonary stenosis and atrial septal defect, have previously been reported in
association with ALDH1A3-associated anophthalmia and microphthalmia (412, 423).
However, as these systemic findings have only been reported in a single individual
each, it still remains unclear if these are associated or unrelated features of ALDH1A3
variation. Occasionally, patients with ALDH1A3-associated anophthalmia and
microphthalmia are also reported to have neurocognitive or behavioural features
including intellectual disability, developmental delay and autism (412, 416, 417, 423).
This association remains controversial due to the wide inter-familial variability in
neurocognitive or behavioural outcomes (416, 417, 423), and the important
confounding impact of visual impairment during development (428, 429). In addition,
intellectual disability due to other genetic disorders may be more common in
populations with high consanguinity (430).
It has previously been suggested that the difference in phenotype between
microphthalmia and anophthalmia may be the result of residual ALDH1A3 activity
(421). However, a review of all known disease-causing mutations in ALDH1A3 (Figure
5.6 and Table 5.6) does not seem to support this hypothesis, with no consistent
correlation between a particular phenotype (anophthalmia or microphthalmia) and the
nature of variation (missense, nonsense, frameshift or splice variants) or the protein
domain affected (NAD-binding domain, catalytic domain or oligomerisation domain).
This may partly be due to difficulties in distinguishing between anophthalmia from
severe microphthalmia in routine clinical practice. True congenital anophthalmia can
only be diagnosed radiologically or histologically, and most published cases of clinical
anophthalmia probably include cases of severe microphthalmia, where residual ocular
tissue may have been present in the orbit despite external appearances of an absent
globe (410). Although systemic features have been reported in ALDH1A3-associated
ocular disease, these are uncommon, and the associations are controversial,
providing a relatively good prognosis for affected families when compared to other
known causes of anophthalmia.
184
Figure 5.6 ALDH1A3 variants associated with anopthalmia and microphthalmia.
(A) Schematic representation of ALDH1A3 NM_000693.3 transcript highlighting the position of all disease-causing variants associated with anophthalmia and microphthalmia identified to date. (B) Schematic showing domain architecture of ALDH1A3 [predicted domains described by Moretti et al (24)] and the locations of all disease-causing variants associated with anophthalmia and microphthalmia identified to date. OD, oligomerisation domain. Variants identified in this study are denoted by a red asterix (*).
To date, there have only been six previously reported variants in TDRD7 associated
with juvenile or congenital cataracts in homozygous form in six families (389, 431-
434), including a further frameshift deletion [c.1129delG; p.(Ala377Profs*2)] in one
other consanguineous Pakistani family (389) (Table 5.7). The identification of a
second TDRD7 variant in Pakistan as part of this study raises the possibility that
TDRD7 variation is a potentially under-recognised cause of congenital cataracts in
Pakistan. Additionally, there have been a small number of paediatric cataract cases
associated with monoallelic TDRD7 variants (431, 435, 436), and there have also been
reports of two families where homozygosity for TDRD7 variants in male individuals
was associated with azoospermia in combination with congenital cataracts (434); the
true causal relationship in these cases however still remains uncertain.
185
Although affected individuals in families 57 and 58 were initially diagnosed with
congenital cataracts, genomic studies subsequently identified homozygous sequence
variants in CYP1B1 and ATOH7 respectively as likely responsible for ocular disease
in these families. Autosomal recessive mutations in CYP1B1 are the most common
cause of primary congenital glaucoma, and the CYP1B1 c.1169G>A; p.(Arg390His)
variant has been found to be particularly common in Pakistani, North Indian, Iranian
and Chinese communities (392, 437-439), supporting a revised clinical diagnosis of
primary congenital glaucoma in this family. The initial misdiagnosis of congenital
cataracts could have been due to difficulties in differentiating between leukocoria
caused by lenticular opacities, and corneal oedema and opacity resulting from raised
intraocular pressures in primary congenital glaucoma.
Disruption of ATOH7 function has been previously described to cause a spectrum of
developmental eye disorders with overlapping phenotypes in only a small number of
families (Table 5.8). Associated conditions include non-syndromic congenital retinal
nonattachment (24, 440, 441), persistent hyperplasia of the primary vitreous (442),
and FEVR (443), with common clinical features including poor vision from birth,
nystagmus, microphthalmia, corneal opacities, cataracts, vitreous anomalies including
persistent foetal vasculature or a retrolental fibrovascular mass, retinal vascular
abnormalities and retinal detachment. ATOH7 variants have also been potentially
implicated in optic nerve hypoplasia and aplasia (444, 445), although the true
association remains unclear (442, 444, 446). Additionally, genome-wide association
studies have implicated ATOH7 variants in association with adult-onset open-angle
glaucoma (447, 448), and a heterozygous ATOH7 deletion has been associated with
primary open angle glaucoma in a single individual (449); however the identification of
two affected individuals (IV:1 and IV:4) in family 58 with features of congenital
glaucoma (Table 5.2) represents the first time ATOH7 variants have been associated
with this phenotype. This is of potential clinical relevance, as in contrast to the
permanent visual field loss associated with glaucomatous optic neuropathy in adults
with adult-onset glaucoma, optic neuropathy in infants and young children with
congenital glaucoma is potentially reversible, especially in the early stages of the
disease (450). Detailed clinical phenotyping in additional families will be helpful to
clarify this potential genotype-phenotype correlation, and a prompt ophthalmic
assessment in all individuals with ATOH7‐associated ocular disease may allow early
186
recognition and appropriate surgical management of congenital glaucoma if present,
significantly improving a child’s visual prognosis.
In family 59, identification of a homozygous LRP5 variant causing global ocular
developmental defects has permitted a refined clinical diagnosis and improved
understanding of disease in affected individuals who did not have a previous
ophthalmic diagnosis. In addition to ocular findings, LRP5 variants are typically
associated with reduced bone mineral density, osteopenia and osteoporosis (451,
452), and appropriate clinical screening of affected individuals may enable early
detection of skeletal co-morbidities and may prevent the development of secondary
complications.
Early onset nystagmus in an otherwise seemingly healthy child can be associated with
a diverse range of ophthalmic conditions including achromatopsia, congenital
stationary night blindness, inherited retinal dystrophies, aniridia and ocular albinism
(144). In family 60, a novel FRMD7 variant was identified as likely responsible for X-
linked congenital idiopathic nystagmus in affected individuals. There are now over 90
FRMD7 variants reported to cause congenital idiopathic nystagmus (453); this is
however the first time FRMD7 has been identified as a cause of congenital idiopathic
nystagmus in Pakistani communities. The FRMD7 molecular diagnosis in this family
provided reassurance for the family regarding the good visual prognosis and non-
progressive nature of the condition, without the need for complex, invasive and
expensive investigations to rule out an underlying ocular disease. In family 61, despite
the consanguineous family structure, identification of a well reported PAX6 nonsense
variant (6, 393-400) occurring de novo in the affected individual allowed accurate
family counselling regarding recurrence risks. Additionally, this provided a molecular
mechanism for the aniridia later identified on re-examination of the affected individual,
and afforded reassurance by excluding a PAX6 deletion extending to the WT1 gene
predisposing to Wilms tumour development (454).
HPS is an autosomal recessive condition where defects in intracellular vesicle
formation and trafficking result in OCA associated with a bleeding diathesis and ceroid-
lipofuscin lysosomal storage disease. This can result in life-threatening manifestations
such as pulmonary fibrosis, granulomatous colitis, and immunodeficiency (405).
187
Awareness of this diagnosis and its associated systemic implications in families 62 -
66 has enabled counselling about the risk of pulmonary fibrosis, and importance of
smoking cessation and prompt treatment of respiratory infections to optimise
pulmonary function. Clinicians caring for the family are also now aware of the risk of
prolonged bleeding and increased need for platelet or red blood cell transfusions
following surgical procedures. The five disease-causing HPS1 variants identified in
this study also now doubles the number of HPS1 variants reported in Pakistani families
(455).
The families investigated in this study highlight the notable diagnostic difficulties
encountered by clinicians in geographically isolated regions in Pakistan, who do not
always have access to the specialised equipment for detailed ocular phenotyping
necessary for an accurate ophthalmic diagnosis. Recognising these genes and
variants as a cause of ocular disease within these communities will facilitate more
accurate clinical diagnoses, early recognition and treatment for individuals with a
similar clinical presentation in the region.
188
Table 5.6 Summary of all reported ALDH1A3 variants associated with anophthalmia and microphthalmia
Type of
variant
Nucleotide
variant
Protein variant Ethnicity Number of
affected
families
(individuals)
Associated
phenotype
Reference ClinVar
(Accession)
Missense c.211G>A p.(Val71Met) Israeli 1 (9) Anophthalmia &
microphthalmia
Mory et al (415) Pathogenic
(VCV000091908)
c.265C>T p.(Arg89Cys) Pakistani 1 (2) Anophthalmia &
microphthalmia
Fares-Taie et al (412) Pathogenic
(VCV000040203)
c.287G>A p.(Arg96His) Chinese 1 (2) Anophthalmia Liu et al (426) Pathogenic
(VCV000978214)
c.434C>T p.(Ala145Val) Saudi Arabian 1 (2) Microphthalmia Aldahmesh et al (416) Not present
c.521G>A p.(Cys174Tyr) Lebanese 1 (3) Anophthalmia &
microphthalmia
Roos et al (417) Not present
c.709G>A p.(Gly237Arg) Chinese &
Iranian
3 (5) Anophthalmia Liu et al (426);
Dehghani et al (418)
Pathogenic
(VCV000978215)
c.845G>C p.(Gly282Ala) Arabic 1 (2) Microphthalmia Alabdullatif et al (419) Not present
c.874G>T p.(Asp292Tyr) Asian British -
Bangladeshi
1 (2) Anophthalmia Patel et al (427) Not present
c.964G>A p.(Val322Met) Indian 1 (1) Anophthalmia Ullah et al (420) Uncertain
significance
(VCV000653567)
c.1064C>G p.(Pro355Arg) Egyptian 1 (1) Anophthalmia Abouzeid et al (421) Not present
c.1105A>T p.(Ile369Pro) Saudi Arabian 1 (3) Microphthalmia Aldahmesh et al (416) Not present
c.1144G>A p.(Gly382Arg) Egyptian 1 (4) Anophthalmia Abouzeid et al (421) Not present
c.1231G>A p.(Glu411Lys) Swiss - Sri
Lankan
1 (1) Microphthalmia Abouzeid et al (421) Not present
189
c.1240G>C p.(Gly414Arg) Pakistani 1 (5) Anophthalmia Lin et al (422) – this
study
Not present
c.1393A>T p.(Ile465Phe) Asian British -
Bangladeshi
1 (2) Anophthalmia Patel et al (427) Not present
c.1398C>A p.(Asn466Lys) Turkish 1 (2) Anophthalmia &
microphthalmia
Semerci et al (423) Not present
c.1477G>C p.(Ala493Pro) Turkish 1 (1) Anophthalmia &
microphthalmia
Fares-Taie et al (412) Pathogenic
(VCV000040204)
Nonsense c.568A>T p.(Lys190*) Egyptian 1 (2) Anophthalmia &
microphthalmia
Yahyavi et al (425) Not present
c.898G>T p.(Glu300*) Swiss -
Spanish
1 (1) Microphthalmia Abouzeid et al (421) Not present
c.1165A>T p.(Lys389*) Hispanic 1 (1) Anophthalmia &
microphthalmia
Yahyavi et al (425) Not present
Splicing c.204+1G>A Alteration of WT
donor site
Egyptian 1 (2) Anophthalmia &
microphthalmia
Abouzeid et al (421) Not present
c.475+1G>T Skipping of exon
5
Moroccan 1 (1) Anophthalmia &
microphthalmia
Fares-Taie et al (412) Pathogenic
(VCV000040205)
c.666G>A Skipping of exon
6
Turkish 1 (7) Anophthalmia &
microphthalmia
Semerci et al (423);
Plaisancié et al (424)
Not present
c.1391+1G>T Alteration of WT
donor site
Egyptian 1 (1) Anophthalmia Abouzeid et al (421) Not present
Frameshift c.1310_1311
delAT
p.(Tyr437Trpfs*4
4)
Pakistani 1 (4) Anophthalmia Ullah et al (420) Not present
c.172dupG p.(Glu58Glyfs*5) Pakistani 1 (3) Anophthalmia Lin et al (422) – this
study
Not present
Abbreviations: WT, wild type. Variants identified in this study are highlighted in yellow
190
Table 5.7 Summary of all reported TDRD7 variants associated with inherited ocular disease
Type of variant Mode of
inheritance
Nucleotide
variant
Protein variant Ethnicity Number of
affected
families
(individuals)
Associated phenotype Reference ClinVar
(Accession)
Missense Monoallelic c.83A>C p.(Gln28Pro) Han
Chinese
1 (1) Bilateral posterior polar
cataract
Li et al (435) Not present
Biallelic
(hom)
c.2539G>A p.(Asp847Asn) Saudi
Arabian
1 (1) AR cataract Alfares et al
(432)
Likely pathogenic
(VCV000800981)
Nonsense Biallelic
(hom)
c.689dupA p.(Tyr230*) Chinese 1 (2) AR congenital cataracts +
azoospermia in males
Tan et al
(434)
Pathogenic
(VCV000427904)
Monoallelic Chinese 1 (1) Cataract
(Also CRYBA2 variant het
carrier)
Sun et al
(436)
Frameshift Biallelic
(hom)
c.1129delG p.(Ala377Profs*2) Pakistani 1 (5) AR congenital cataracts Chen et al
(389)
Pathogenic
(VCV000426069)
Biallelic
(hom)
c.2469delG p.(Asn824Thrfs*27) Pakistani 1 (2) AR congenital cataracts This study Not present
Biallelic
(hom)
c.328dupA p.(Thr110Asnfs*30) Chinese 1 (3) AR congenital cataracts +
azoospermia in males
Tan et al
(434)
Pathogenic
(VCV000427905)
Inframe deletion Biallelic
(hom)
c.1852_1854
delGGT
p.(Val618del) NA 1 (4) AR congenital cataracts Lachke et al
(431)
Not present
Biallelic
(hom)
NA 1 (1) NA Maddirevula
et al (433)
Chr 9 paracentric
inversion
disrupting TDRD7
& NR5A1
Monoallelic 46,X,Y,inv(9)(q22.33q34.11) NA 1 (1) Juvenile cataract and
hypospadias
(NR5A1 disruption thought
responsible for hypospadias)
Lachke et al
(431)
-
Abbreviations: AR, autosomal recessive; het, heterozygous; hom, homozygous. Variant identified in this study is highlighted in yellow.
191
Table 5.8 Summary of all reported biallelic ATOH7 variants associated with inherited developmental ocular disease
Type of variant Nucleotide
variant
Protein variant Ethnicity Number of
affected
families
(individuals)
Associated phenotype Reference ClinVar
(Accession)
Missense c.125G>C p.(Arg42Pro) Pakistani 1 Non-syndromic congenital retinal
nonattachment
Keser et al (440) Not present
c.136A>C p.(Asn46His) Pakistani 1 (3) Persistent hyperplasia of the
primary vitreous
Prasov et al (442) Pathogenic
(VCV000144067)
c.146A>T p.(Glu49Val) Pakistani 1 (5) Ocular developmental defect:
features include nystagmus,
reduced vision, dense corneal
opacity, microphthalmia,
microcornea, retrolental mass
Khan et al (443) Pathogenic
(VCV000144065)
c.175G>A p.(Ala59Thr) Swiss 1 (2) Optic nerve hypoplasia, foveal
hypoplasia and vascular
abnormalities
Atac et al (444) Pathogenic
(VCV000812672)
c.176C>T p.(Ala59Val) Swiss 1 (2) Optic nerve hypoplasia, foveal
hypoplasia and vascular
abnormalities
Atac et al (444) Uncertain
significance
(VCV000812673)
Frameshift c.53delC p.(Pro18Argfs*69) Turkey 1 (2) Familial exudative
vitreoretinopathy
Khan et al (443) Pathogenic
(VCV000144066)
c.94delG p.(Ala32Profs*55) Pakistani 1 (5) Global ocular developmental
defect: features include cataracts,
congenital glaucoma, retinal
detachment
This study Not present
192
Inframe deletion c.121_144del24 p.(Arg41_Arg48del) Japanese 1(1) Non-syndromic congenital retinal
nonattachment
Kondo et al (24) Uncertain
significance
(VCV001034468)
6523bp deletion
in 5’UTR
c.-22208_-
15686del6523
Deletion of
transcriptional
enhancer
Kurdish 1 (4) Non-syndromic congenital retinal
nonattachment
Ghiasvand et al
(441)
Pathogenic
(VCV000030807)
Pakistani 2 (2) Non-syndromic congenital retinal
nonattachment
Keser et al (440)
Abbreviations: hom, homozygous. Variant identified in this study is highlighted in yellow
193
5.4 Conclusions and future work
Studies of families with inherited ocular diseases in Pakistani and Palestinian
communities have helped clarify the phenotypic characterisation and genetic
associations of rare and ultra-rare inherited eye diseases, and provide much-
needed scientific insight into the spectrum, nature and causes of ocular disease
within these communities and globally.
Mitochondrial complex II deficiency is a rare inborn error of metabolism,
accounting for approximately 2% of mitochondrial disease diagnoses. Only 61
patients have been described to date, associated with pathogenic variants in
four complex II genes (SDHA, SDHB, SDHD and SDHAF1); of these, only two
affected individuals have been reported with candidate biallelic variants in the
SDHD gene (456). The three additional affected individuals described in this
study further consolidate recessive SDHD variants as contributing to
mitochondrial complex II deficiency. There is unfortunately no cure for complex
II deficiency, although some therapeutic interventions may be associated with
symptomatic improvements in these patients, including riboflavin, l-carnitine
and ubiquinone (456). Further studies integrating detailed clinical phenotyping
and metabolic investigations, molecular genomic analyses combined with in
silico pathogenicity prediction and structural modelling, histological and
histochemical studies of muscle biopsy specimens, spectrophotometric
assays of OXPHOS respiratory chain function, BN-PAGE and SDS-PAGE
followed by Western blot analysis to study physiological mitochondrial complex
protein-protein interactions, and complementation studies in patient and yeast
cells will be useful in developing a better understanding of the molecular basis
of these conditions, which may lead to the development of more effective
therapies.
TDRD7 (389, 431-434) and ATOH7 (24, 440-443) are uncommon causes of
developmental eye disorders. ATOH7 dysfunction is associated with a wide
spectrum of developmental ocular phenotypes including non-syndromic
congenital retinal nonattachment (24, 440, 441), persistent hyperplasia of the
194
primary vitreous (442), and FEVR (443), and potentially implicated in optic
nerve hypoplasia and aplasia (444, 445). ATOH7 is known to function as an
early retinal transcription factor crucial for RGC development and specification
(441), and further functional characterisation in animal models or iPSC-derived
retinal organoids may be useful in assessing the impact of pathogenic ATOH7
variants on RGC morphology, and delineate the mechanisms by which ATOH7
dysfunction leads to the diverse developmental ocular phenotypes described.
TDRD7 is a component of ribonucleoprotein complexes and RNA granules in
differentiating lens cells, suggesting that post-transcriptional regulation of gene
expression plays an important role in lens homeostasis (431). TDRD gene
polymorphisms have potentially been associated with susceptibility to age-
related cataracts (457), and there is also increasing evidence for additional
genes implicated in the development of both congenital and adult-onset
cataracts, such as LIM2, EPHA2, and CRYAA (458, 459). Understanding the
genetic basis and molecular pathogenesis of congenital cataracts may lead to
the development of strategies that prevent or delay the development of age-
related cataracts, hence reducing the enormous global burden for cataract
surgery. Studies of RNA and protein-binding targets of TDRD7, coupled with
bioinformatics strategies, have identified several potential interacting partners
of TDRD7, including HSPB1, NRAS and ACTN2 (460-462). Further
investigation into these molecular pathways, including subcellular localisation
studies in lens fiber and epithelial cells, characterising gene expression in the
lens during development, and studying ocular and lens development in
xenopus, zebrafish or mouse gene-perturbation models (463-465), may yield
new insights into cataractogenesis.
There is a wide phenotypic variation in ALDH1A3-associated ocular disease.
Individuals with the same ALDH1A3 variant can display both anophthalmia and
microphthalmia in different eyes (419, 421, 425), and affected individuals with
the same variant within the same family have been found to have clinical
phenotypes of differing severity (417, 421, 423-425). Epidemiological studies
have predicted the contribution of both genetic and environmental factors in the
pathogenesis of congenital eye defects including anophthalmia and
195
microphthalmia (466), and the wide phenotypic spectrum seen may result from
the impact of modifying genes or environmental influences affecting the
ALDH1A3-associated eye disease phenotype. Further studies including DNA-
methylation studies in animal models investigating epigenomic influences,
transcriptomic analyses in patients linking altered gene expression to
differential phenotypic outcomes, and co-immunoprecipitation and pull-down
assays to investigate ALDH1A3 protein interactions, would be useful to define
this interaction and elucidate underlying pathways. Given that ALDH1A3 gene
mutations appear to be the most common cause of anophthalmia and
microphthalmia in consanguineous families (412, 415-425), screening for
variants in this gene before exome analysis in populations with high rates of
consanguinity can be considered.
196
6 CONCLUDING COMMENTS
This thesis documents the results of clinical and genetic analyses in a total of
66 Amish, Pakistani and Palestinian families, with a range of genetically
undiagnosed syndromic and non-syndromic inherited ocular diseases. The
work described in this thesis entails the identification of 18 novel pathogenic
variants in 10 genes associated with a variety of inherited ocular disorders,
including OCA (TYR, OCA2, HPS1), congenital nystagmus (FRMD7), ocular
developmental disorders (ALDH1A3, TDRD7, ATOH7 and LRP5) and
ciliopathies (BBS5, SCAPER). This knowledge significantly expands on the
molecular spectrum of inherited eye diseases in communities and globally. In
many families investigated, the molecular diagnosis enabled a subsequent
confirmation or refinement of the initial clinical diagnosis, as detailed in chapter
5.3, with important treatment and prognostic implications for affected individuals
and their families.
In the Amish, Pakistani and Palestinian communities, geographical isolation,
common ancestry and endogamy, combined with the often large family sizes
typical of families in these regions, often results in an enrichment of disease-
causing founder variants. These often represent important causes of disease
in a particular region, and identification of the INPP5E c.1879C>T; p.(Gln627*)
founder variant associated with MORM syndrome in the Punjab region in
Pakistan (described in chapter 4.2), as well as the SCAPER c.2236dupA,
p.(Ile746Asnfs*6) founder variant in the Ohio Amish community (described in
chapter 4.4), allows targeted genetic testing for these variants, permitting a
more rapid and cost-effective means of achieving a molecular diagnosis,
shortening the diagnostic odyssey for affected individuals within the community.
There is a relative lack of knowledge regarding the specific nature and causes
of inherited ocular diseases particularly in developing nations such as Pakistan.
Literature reviews were conducted, compiling the most comprehensive and
curated reports of genes and variants associated with BBS and OCA in
Pakistan to date (presented in Appendices B and C respectively). This work
197
highlights the OCA2 c.1045-15T>G and p.(Asp486Tyr) founder variants, as well
as the TYR p.(Arg278*) recurrent variant as contributing significantly to OCA in
Pakistan. These reviews will enhance knowledge of the genomic architecture
pertaining to these inherited eye diseases in Pakistan, and facilitate the
development of community-relevant disease databases correlating allele
frequencies with geographical localisation and ethnicity. This is of enormous
value for local healthcare resource planning, enabling the design of community-
specific hierarchical strategies for rapid and cost-effective genetic testing arrays
to enable an accurate disease diagnosis to be achieved more rapidly, thus
aiding the development of diagnostic and clinical care pathways and policies
throughout Pakistan.
This thesis also details comprehensive clinical and genetic studies of a number
of rare inherited ocular disorders, described in only small numbers of affected
individuals to date. This includes MORM syndrome and SCAPER syndrome
(described in chapters 4.2 and 4.4), mitochondrial complex II deficiency
associated with biallelic SDHD variation (chapter 5.2), TDRD7-associated
congenital cataracts (chapter 5.3), and developmental eye defects associated
with ALDH1A3 and ATOH7 (chapter 5.3). The deep phenotypic characterisation
performed in these study families allowed a more comprehensive clinical
comparison to be made with the previously reported cases, expanding on the
disease-associated clinical spectrum, delineating the core and variable
phenotypic features, and clarifying the presence (or indeed absence) of any
genotype-phenotype correlation associated with variants in these genes.
The work of this thesis also provides novel insights into known inherited eye
disease genes. The role of two common TYR variants p.(Ser192Tyr) and
p.(Arg402Gln) in OCA, previously considered to represent non-pathogenic
polymorphic alleles although subsequently contested, has been debated over
the last 10 years. Chapter 3.2 now provides strong evidence that the TYR
p.(Ser192Tyr)/p.(Arg402Gln) in cis haplotype may cause disease both when
occurring in compound heterozygous fashion with a second deleterious allele,
or in doubly homozygous state. The increased frequency of the BBS1
p.(Met390Arg) variant in the Pennsylvania Amish community will similarly
198
permit empowered genetic and co-segregation studies able to determine
phenotypic penetrance of this hypomorphic variant, which has important
diagnostic implications given that this common founder variant is responsible
for ~80% of BBS1 cases in European populations (297). Alongside detailed
phenotypic assessments, this will allow clarity into the potential contribution of
any additional BBS modifier variants, and may provide evidence supporting or
disputing the controversial triallelic hypothesis in BBS. To this end, recruitment
of additional family members in the extended Amish family with BBS studied in
chapter 4.3 is ongoing, and detailed phenotype information is being obtained in
collaboration with local ophthalmologists and clinicians.
Within this thesis, several sequencing techniques were employed, often in a
hierarchical fashion, in the genomic investigation of families with inherited eye
diseases. For instance, albinism families often first underwent TYR gene
screening, followed by Illumina TruSightTM One clinical exome sequencing, in
order to detect causative variants in known albinism genes, whilst
consanguineous families with complex ocular phenotypes were instead
investigated with genome-wide SNP genotyping and WES in the first instance,
which would facilitate the detection of rare or even novel disease genes
involved in hereditary eye diseases. Each genomic technique has recognised
limitations and advantages, balancing a more comprehensive analysis against
the difficulties in interpreting large numbers of variants; and the tailored
approach adopted in this study enabled a pragmatic, efficient and cost-effective
means of achieving a molecular diagnosis in many affected families, reflecting
the likely real-word application of genomic technologies in these resource-
scarce communities.
Studies in this thesis have contributed to a recent development of a new
microarray platform diagnostic testing panel, led by the WoH project and now
available in the Wisconsin Clinical Laboratory Improvement Amendments
(CLIA) - certified regional testing laboratory. This flexible platform offers a rapid
and cost-effective means of targeting the specific gene mutations of >150
genetic disorders identified across the Amish communities, and can be readily
expanded to incorporate new disease-associated variants as they are identified
199
within the community. Testing via the platform is offered at cost to the Amish
community, and has particular utility as part of a molecular-based newborn
screening program, allowing affected individuals and families to benefit from
early diagnosis and intervention. One particular benefit will be in the early
diagnosis of propionic acidemia, an inborn error of metabolism occurring at an
increased frequency in the Amish-Mennonite communities due to a founder
missense variant in PCCB [c.1606A>G; p.(Asn536Asp)] (467). This is a
condition where early diagnosis and intervention is shown to improve long-term
neurological outcomes (468), however, cases may be missed using standard
newborn screening metabolic assays. This genetic testing platform may also be
of use as a carrier screening assay as the communities grow more receptive
towards this, permitting anticipatory health care to prevent or mitigate the
devastating effects of genetic diseases that may occur in the newborn.
In recent years, advances in NGS technologies and bioinformatics processing
algorithms, tools, and pipelines, have led to improvements in the diagnosis of
Mendelian disorders. Despite this, overall diagnostic rates for exome
sequencing, the primary genomic technique employed in this study, is limited
to ~25 - 50% (433). Some of these molecularly undiagnosed individuals may
harbour variants in genes yet to be associated with human disease. Studies of
inherited eye diseases in genetically isolated populations such as the Amish
communities of the USA, or rural Pakistani and Palestinian communities, where
the unique genomic architecture of these communities enables the application
of empowered genomic approaches for disease gene identification (60, 469,
470), together with genomic matchmaking initiatives (471), will facilitate the
identification of novel gene-disease associations, whilst also providing
desperately required healthcare benefits for the families and populations
involved.
The relatively low diagnostic rate of exome sequencing may also reflect the
restricted focus, limited to coding sequences and intronic sequences flanking
the exon-intron boundaries, representing only ~1.5% of the human genome.
The application of whole genome sequencing approaches, which enable the
detection of structural variants, deep intronic and regulatory variants in non-
200
coding regions, may lead to further improvements in diagnostic yield, and is an
option being considered for any remaining molecularly undiagnosed individuals
recruited through the established collaborative research consortium involving
the community genomics research group at the University of Exeter, and
collaborating scientists and clinicians within the Amish communities and at
research centres in Pakistan and Palestine.
Interestingly, it has been suggested that genome sequencing only offers a
minimal uplift in diagnostic yield compared to exome sequencing, and that the
primary limitation to molecular diagnostic rates may relate not to the capture or
calling of causal variants at the sequencing stage, but rather in the interpretation
of the genomic variants identified (433). Current strategies for variant
interpretation include family segregation, computational variant effect
prediction, and functional assays (472), and are fairly reliable when applied to
protein-coding regions of the genome, although predicting the pathogenicity of
missense variants can be challenging (473), as demonstrated by the uncertain
significance of the novel OCA2 p.(Arg588Trp) variant identified in family 38
(chapter 3.3). Other sequence variants, such as deep intronic, enhancer or
promoter region variants, as well as coding sequence variants with splice-
disrupting potential, remain difficult to interpret, despite their clear contribution
to Mendelian disease. Whole transcriptome sequencing approaches (RNA-seq)
allowing quantification of gene expression levels and detection of alternative
splicing (474), as well as multiplexed functional assays for variant effect (475),
show promising potential in empowering variant interpretation and resolving the
variants of uncertain significance “trapped in the interpretative void between
benign and pathogenic” (476). These approaches are currently largely
restricted to research environments, but their validation and integration into
mainstream clinical practice may bring about transformative benefits in rare
disease diagnostics.
Together, the work presented in this thesis provides an improved knowledge of
the clinical and molecular spectrum of hereditary eye diseases in the Amish,
Pakistan and Palestinian communities. These findings enabled targeted
changes in clinical management and accurate prognostic information to be
201
provided to affected individuals and families, with communities benefitting from
targeted genetic testing strategies that permit cost-effective disease diagnosis
and improved carrier detection. This new knowledge may also enable
experimental approaches for restoration of protein function through gene
therapy, such as Luxturna® (voretigene neparvovec), the first ever licensed
gene replacement therapy for the treatment of a genetic disease in humans,
approved for the treatment of patients with RPE65-associated retinal dystrophy
(12). In this way, an improved understanding of disease pathways arising from
the discovery of novel disease genes identified in a community setting may thus
ultimately also be applied to genetically diverse populations such as the UK and
bring about clinical benefits for patients worldwide.
202
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561. Morgan NV, Pasha S, Johnson CA, Ainsworth JR, Eady RA, Dawood B, et al. A germline mutation in BLOC1S3/reduced pigmentation causes a novel variant of Hermansky-Pudlak syndrome (HPS8). The American Journal of Human Genetics. 2006;78(1):160-6.
562. Badolato R, Prandini A, Caracciolo S, Colombo F, Tabellini G, Giacomelli M, et al. Exome sequencing reveals a pallidin mutation in a Hermansky-Pudlak-like primary immunodeficiency syndrome. Blood. 2012;119(13):3185-7.
247
APPENDIX A: NGS AND AUTOZYGOSITY MAPPING
A.1 NGS
The traditional approach to disease gene identification in Mendelian diseases involved
dideoxy sequencing of candidate genes selected based on their predicted relevance
to disease pathophysiology, their location within a disease locus identified through
positional mapping, or a combination of both. This approach however is reliant on prior
knowledge of the predicted protein structure or function and molecular pathway of
disease.
The advent, widespread availability and decreasing costs of high throughput NGS
technologies now provide an efficient and cost-effective approach for the unbiased
interrogation of an individual’s genome. This includes exome sequencing, which
investigates the protein-coding regions (exons and splice sites) of the genome, where
approximately 85% of Mendelian disease-causing variants are found (477), as well as
genome sequencing, which will also allow the detection of variants in non-coding and
regulatory genomic regions.
NGS approaches have dramatically accelerated the discovery of novel disease genes
for rare Mendelian disorders (478) and have a transformative potential in disease
diagnosis and personalised medicine, with genomic medicine initiatives currently
being integrated into healthcare systems across the world (86). NGS approaches
however often result in large numbers of genomic variants identified [over 20,000
variants in each exome (479) and several million in each genome (480)], and the
challenge therefore now shifts from disease gene identification to variant
interpretation. This has been aided by the development of population variant
databases such as the gnomAD (481), disease variant databases such as ClinVar
(482) and HGMD (483), and in silico variant pathogenicity prediction tools such as
SIFT (484), Polyphen-2 (485) and MaxEntScan (486).
There is however still a need for robust and efficient variant prioritisation strategies to
identify the pathogenic variant and prove disease causality. One such strategy
248
combines a gene sequencing method such as exome or genome sequencing with a
genome scanning method such as autozygosity mapping, allowing the rapid
identification of potential disease-causing sequence alterations within a disease locus
that can be prioritised for further investigation (44).
A.2 Autozygosity mapping
Autozygosity mapping is a powerful technique used to identify candidate loci for
autosomal recessive disease genes in populations where there is a significant founder
effect due to unions between closely related (consanguineous) or more distantly
related individuals (such as endogamous populations with a high frequency of inter-
community marriage). This approach relies on the principle that affected individuals in
these populations will be homozygous for the same disease variant within a genomic
segment or haplotype inherited from a common ancestor, referred to as autozygosity
or ‘identity by descent’ (487). The genome is scanned to identify or ‘map’ the
autozygous regions shared exclusively and consistently by affected individuals,
signposting the location where the pathogenic variant is likely to be discovered (Figure
A1).
Figure A1 Principles of autozygosity mapping
Red boxes across chromosomes indicate regions of homozygosity in affected individual, with the common autozygous region shared across all affected individuals indicating the most probable location of the disease-causing gene. Image created with BioRender.
Historically, autozygosity mapping was dependent on the analysis of microsatellite
markers, however, this has largely been superseded by SNP genotyping panels and
microarray technology. SNPs, being diallelic, are less informative than the highly
polymorphic microsatellites, but are dispersed more equally throughout the genome
249
at a higher density, allowing the detection of smaller autozygous regions (488).
Additionally, SNP genotyping platforms are easily automated, permitting higher
throughput and lower costs, and can also be used to detect CNV (489).
There are important pitfalls to be aware of in the interpretation of autozygosity mapping
results, particularly in the study of rare diseases in community settings. Successful
identification of disease loci using this technique relies on correct ascertainment of
affectation status in family members for accurate inheritance models and interpretation
of results. In ophthalmology, this often relies on specialist equipment and expertise
that is usually based in the larger teaching hospitals, whilst affected families in
communities often reside in more rural and geographically isolated locations with
limited accessibility. Additionally, given the enrichment of disease-causing mutations
within communities due to a founder effect, a situation may arise where a disease may
be caused by compound heterozygous mutations (490, 491), or two phenotypically
similar conditions may segregate independently in a single family (especially taking
into account the clinical and genetic heterogeneity inherent in many inherited eye
diseases) (331, 491, 492), further complicating the interpretation of results generated
using autozygosity mapping techniques. Despite these considerations, the combined
approach of autozygosity mapping with NGS can helpful in clarifying molecular
diagnoses in conditions that are diagnostically challenging due to genetic and
phenotypic heterogeneity, such as inherited retinal dystrophies (493, 494).
With the increasingly widespread use of exome and more recently genome
sequencing in both diagnostic and research environments, the large volume of
sequence data information generated by these NGS technologies raises the possibility
of analysing the exome or genome sequence data itself to simultaneously map
autozygous regions (without the need for the genome-wide SNP-genotyping
microarrays that were previously used for this purpose) and concurrently identify all
potentially deleterious sequence variants within these candidate disease loci. This
one-step experimental approach is attractive in principle, and several bioinformatic
tools have been developed for this purpose (495-497). However, these analyses do
have their limitations; with exome data, the coverage can be poor with numerous gaps
due to the uneven distribution of coding regions across the genome, and there is the
potential for shorter autozygous regions located within gene-poor regions to be missed
250
(495), whilst the lower read depth of genome sequencing can result in erroneous SNP
calls due to errors in sequencing or chance sampling of only one allele at a genuinely
heterozygous position (498). With an awareness of these limitations however,
adopting a cautious approach to analysis with a greater level of thought and subjective
data interpretation, the use of exome or genome data for detecting regions of
autozygosity can still be an effective adjunct in the detection of novel disease genes,
and for rapid screening for deleterious variants in known disease genes.
A.3 Advances in sequencing technologies
Whilst NGS technologies have facilitated significant advances in medical research,
they are not without their limitations. NGS methods are reliant on PCR, which is
inefficient in amplifying genomic regions with a high GC content (499). NGS platforms
are also dependent on clonal amplification to create clusters of molecules, with the
potential for base incorporation errors in individual molecules within a cluster, thereby
increasing the noise within samples, and limiting the reads to short lengths of 50-500
bp (500). Numerous short reads require extensive assembly, and genomes often
contain repeat sequences that are longer than the NGS reads, resulting in
complexities in alignment that can lead to mis-assemblies and gaps (501). Some areas
of the genome, such as areas of repetitive DNA, high sequence homology, or even
extreme GC content, are therefore still difficult to analyse with current NGS platforms
(500).
There are further limitations to the use of NGS technologies for variant analysis in rare
diseases. Whilst short reads allow accurate detection of smaller variants such as
single-nucleotide variations and short indels, the detection and characterisation of
larger structural variants is challenging, and there is limited capacity to link
independent variants on the same gene for phasing of alleles and discrimination of
parental origin, which is important in situations where compound heterozygosity is
identified (233).
The advent of third-generation sequencing, also known as long-read sequencing, may
provide a solution to the aforementioned issues. Third-generation sequencing
platforms work by single-molecule sequencing in real time with no amplification,
251
directly providing long linear read lengths (1 – 100 kb) and fast sequencing times (2-
10 hours) (500). Two commercially available third-generation sequencing
technologies include the single-molecule real-time (SMRT) sequencing by Pacific
Biosciences (PacBio) and nanopore sequencing by Oxford Nanopore Technologies
(ONT). PacBio utilises a sequencing-by-synthesis approach, with real-time acquisition
of signals from fluorescently-labelled nucleotides as they are being incorporated into
single DNA molecules (502). Nanopore sequencing instead identifies nucleotides by
measuring their electrical conductivity as they pass through the nanopore membrane
(503). Both techniques have drawbacks compared to standard NGS platforms,
including higher sequencing error rates, lower throughput and higher costs (504), and
developments to address these will be needed before these third-generation
sequencing technologies can be widely adopted in clinical diagnostic and research
settings.
There is also a need for additional biological knowledge that can highlight the
molecular signature of the disease. This can aid in the identification of candidate
disease genes and variants, particularly for non-coding and regulatory variants, and
enable a better understanding of their disease mechanism. One potential route is
through functional genomic assays via broad-based ‘omics’ approaches, which study
the impact on different cellular products including gene transcripts (transcriptomics),
proteins (proteomics) or metabolites (metabolomics), in affected individuals compared
to unaffected controls, allowing the identification of aberrant cellular products or
activities. One such technique is RNA-sequencing (RNA-seq), which measures
transcript levels and diversity in individual tissue types (505), and can be used to
highlight the consequences of both coding and non-coding variants on gene
expression levels or alternative splicing. Recent applications of this technique have
aided in the diagnosis of several rare diseases, with particular success for conditions
with a neuromuscular or mitochondrial phenotype (506, 507).
Recent advances in sequencing technologies and bioinformatics have greatly
improved our diagnosis and understanding of rare Mendelian disorders. Future efforts
are likely to focus on combining the current and developing technologies into a single
streamlined workflow for disease variant identification, prioritisation and
252
characterisation, which will hopefully improve the diagnostic yield in monogenic
disorders and shed further light on the complexities of oligogenic diseases.
A.4 Glossary of terms
ClinVar: This is a freely accessible public database that archives and aggregates
reports with supporting evidence about relationships between human variation,
phenotypes and clinical significance (482).
Genome Aggregation Database (gnomAD): This database was developed by an
international coalition of investigators, and aggregates and harmonises both exome
and genome sequencing data from a wide variety of large-scale sequencing projects
(481). The v2.1.1 dataset (GRCh37) contains 125,748 exome sequences and 15,708
whole-genome sequences from unrelated individuals sequenced as part of various
disease-specific and population genetic studies. The v3.1.1 dataset (GRCh38) spans
76,156 genomes as selected in v2.
Human Gene Mutation Database (HGMD): This is a database collating germ-line
variants in nuclear genes reported in the literature in association with human inherited
diseases, and includes both disease-causing variants as well as functional
polymorphisms (483).
Minor allele frequency (MAF): the frequency of the alternative allele(s) in a given
population.
Mapping quality (MQ): This is the root mean square of the mapping quality of reads
across all reads at the site, and provides an estimation of the overall mapping quality
of reads supporting a variant call.
MaxEntScan: This in silico algorithm is based on the ‘maximum entropy principle’
(486). The splice signal given by a wild-type sequence is compared to the splice site
signal given by a mutated sequence, and a change in splice site signal of ≥10% is
considered to predict a pathogenic effect.
253
Polymorphism Phenotyping v2 (Polyphen-2): This in silico algorithm applies to non-
synonymous variants, and predicts the functional significance of an amino acid
substitution using sequence-based and structure-based predictive features (485).
Scores range from 0.0 (tolerated) to 1.0 (deleterious).
Protein Variation Effect Analyser (PROVEAN): This in silico algorithm applies to non-
synonymous and indel variants, and predicts whether an amino acid substitution or
indel has an impact on the biological function of a protein based on the degree of
conservation of amino acid residues in sequence alignments derived from closely-
related sequences (508). If the generated score is ≤ -2.5, the protein variant is
predicted to have a deleterious effect; if the score is above the threshold, the variant
is predicted to have a neutral effect.
Read depth (DP): This is the number of reads that have passed the variant caller’s
internal quality control metrics.
Sorting Intolerant From Tolerant (SIFT): This in silico algorithm applies to non-
synonymous variants, and predicts whether an amino acid substitution affects protein
function based on sequence homology and the physical properties of amino acids
(484). Scores range from 0.0 (deleterious) to 1.0 (tolerated).
SpliceSiteFinder-like (SSF): This prediction program uses an algorithm based on
Shapiro and Senapathy et al (509) using position weight matrices computed from a
set of human constitutive exon/intron junctions for donor and acceptor sites. The splice
signal given by the wild-type sequence is compared to the splice site signal given by
the mutated sequence. A change in splice site signal of ≥10% is considered to predict
a pathogenic effect.
Splice Site Prediction by Neural Network (NNSplice): This prediction algorithm is
based on neural networks, and analyses the structure of donor and acceptor sites
using a separate neural network recogniser for each site (510).
254
APPENDIX B: A NOVEL BBS5 VARIANT ASSOCIATED WITH
BBS IN A PAKISTANI FAMILY
Clinical and genomic investigations were undertaken in a Pakistani family (family 42)
where affected individuals displayed phenotypic features that were highly suggestive
of BBS, in order to aid diagnosis and clinical management.
B.1 Materials and methods
Family recruitment, clinical assessment, blood sample collection and DNA extraction
was performed as previously described (see section 4.2.2). WES was undertaken
using DNA from a single affected individual in family 42 (individual IV:1) at BGI Hong
Kong, as described in section 2.3.5. Bioinformatic analysis with additional virtual gene
panel analysis and filtering to retain heterozygous variants compatible with triallelism
was performed as described in section 4.2.2. Primer design, PCR and dideoxy
sequencing (Appendix Table D2) was also performed as previously described in
section 2.3.3 to genotype and confirm appropriate segregation of the candidate
disease variant in all available affected and unaffected individuals.
A literature review was performed as described in section 2.4 to retrieve all BBS-
associated variants reported in Pakistani families. Findings are summarised in Table
B2 and Figure B2.
B.2 Results: clinical and genetic findings
A multigenerational family (family 42) with two affected individuals (IV:1 and IV:4)
residing in a remote village in the KPK province of Pakistan was investigated. Both
affected individuals were described to have moderate developmental delay/intellectual
disability, visual impairment, truncal obesity, postaxial polydactyly, and renal
anomalies. A single affected male was also reported to have hypogonadism (individual
IV:1) (Table B1).
255
Table B1 Summary of clinical features observed in affected individuals in family 42 with BBS and homozygous for the BBS5 c.196delA variant
Abbreviations: F, female; M, male; yrs, years; (S), severe/profound; (M), mild/moderate. The
(✓) and (✗) symbols indicate the presence of absence of a feature in an affected subject
respectively.
After filtering of variants for zygosity, call quality, population frequency and predicted
outcome in WES data in a single affected individual (IV:1), only a single plausible
candidate disease variant of potential relevance to the phenotype was identified, a
homozygous novel BBS5 frameshift variant (GRCh38) chr2:g.169487122delA;
NM_152384.2:c.196delA; p.(Arg66Glufs*12). This variant was absent in gnomAD
(v2.1.1 and v3.1.1) and from a control exome dataset of 100 ethnically matched
controls undertaken in the Human Molecular Genetics laboratory in Pakistan. This
variant segregated appropriately for an autosomal recessive condition in the family
(Figure B1).
Additional analysis of exome data undertaken using the “rare multisystem ciliopathy
disorders v1.84” PanelApp virtual gene panel did not identify any homozygous or
compound heterozygous candidate variants compatible with the phenotype. No
heterozygous variants compatible with triallelism were identified.
256
Figure B1 Family 42 pedigree showing BBS5 c.196delA genotype data
(A) Pedigree diagram of family 42 showing segregation of the BBS5 c.196delA; p.(Arg66Glufs*12) variant, co-segregating appropriately for an autosomal recessive condition.. Genotypes are shown beneath generations III and IV (+, c.196delA; -, wild type). (B) Sequence chromatogram of BBS5 c.196delA in a homozygous affected individual. (C) Schematic showing domain architecture of BBS5 [adapted from Nachury et al (287) and the location of the c.196delA; p.(Arg66Glufs*12) variant. This variant is located within the first of two predicted Pleckstrin Homology-like (PH-like) domains (PH-B1 and PH-B2).
B.3 Discussion BBS5, located on chromosome 2q31.1, encodes the BBS5 protein, one of eight
proteins that form the stable core of the BBSome protein complex. BBS5 is structurally
and functionally unique among the BBSome components in that it has two Pleckstrin
Homology-like (PH-like) domains that are capable of binding to phosphoinositides
(Figure B1), with this interaction predicted to play a critical role in ciliogenesis (287).
Bbs5 knockout or mutant mice develop a complex phenotype consisting of increased
pre-weaning lethality, craniofacial and skeletal defects, ventriculomegaly, pituitary
anomalies, obesity, retinal degeneration and male infertility; recapitulating features
previous described in ciliopathy syndromes (511, 512).
Here, WES analysis identified a novel homozygous variant in BBS5, confirming a
diagnosis of BBS as likely underlying the clinical features in the affected family (family
42) investigated. This BBS5 c.196delA variant is predicted to result in a frameshift
[p.(Arg66fsGlufs*12)] and nonsense‐mediated mRNA decay. The two affected
individuals demonstrate classical features of BBS, including postaxial polydactyly,
257
learning difficulties, retinitis pigmentosa, and truncal obesity (284). Intra-familial
phenotypic variability was noted, with individual IV:1 displaying more significant visual
impairment and intellectual disability compared to his affected sister. Bilateral
postaxial polydactyly of the lower limbs and clinodactyly of the hands was identified
on examination in individual IV:1, whereas his sister IV:4 had bilateral postaxial
polydactyly in both upper and lower limbs and short toes. This intra-familial phenotypic
variability is well described in BBS, and “triallelism” has been proposed as a possible
mechanism for this, where affected individuals carry homozygous or compound
heterozygous variants at one BBS locus with a third heterozygous variant in a second
BBS gene that interacts to cause or modify the disease phenotype (254). However,
virtual gene panel analysis of BBS genes undertaken in the present study did not
identify any candidate variants that could plausibly be compatible with triallelism.
In populations of Northern European descent, BBS1 and BBS10 are the most
commonly mutated genes, responsible for approximately 23% and 20% of BBS cases
respectively (300, 513), with the BBS1 p.(Met390Arg) (297) and BBS10
p.(Cys91fsLeufs*5) variants (307) being the most common pathogenic alleles for each
of the two genes. In Pakistan, variants in ten out of the 21 BBS-associated genes have
been identified in 74 affected individuals from 28 BBS families (Table B2) (61, 80, 511,
514-528), including two individuals from the current study family (Figure B2). Although
ARL6 and TTC8 are minor contributors to BBS globally, within the Pakistani
community, they account 13.3% and 12.0% of all BBS cases respectively, and are the
two most commonly mutated genes associated with BBS in Pakistan (Figure B2).
To date, there have only been two BBS5 variants associated with BBS in Pakistani
families: a homozygous missense variant of the start codon [c.2T>A; p.(M1?)] likely
resulting in either failure of protein translation or in the translation of an illegitimate
transcript reported in two families (520), and a 11‐bp deletion [c.743_744del11;
p.(Glu245Glyfs*18)] frameshift mutation likely resulting in loss of function in a further
two families (524) (Table B2). The novel BBS5 variant identified in this study therefore
significantly expands on the contribution of BBS5 mutations to BBS in Pakistan.
258
Figure B2 Contribution of BBS genes to BBS globally and within Pakistani families
Global figures derived from (529)
259
Table B2 Summary of all reported variants associated with BBS in Pakistan
Gene Nucleotide variant Protein variant Number of
reported
families
(individuals)
Region in
Pakistan
Reference ClinVar
(Accession)
BBS1
(NM_024649.5)
c.47+1G>T Affects splicing 1 (2) Central Punjab Ajmal et al (515) Not present
c.442G>A p.(Asp148Asn) 1 (2) Central Punjab Ajmal et al (515) Likely pathogenic
(VCV000645579)
c.887delT p.(Ile296Thrfs*7) 1 (1) NA Billingsley et al (517) Pathogenic
(VCV000193740)
BBS2 c.443A>T p.(Asn148Ile) 1 (2) Bagh, AJ&K Ali et al (516) Not present
(NM_031885.5) c.1237C>T p.(Arg413*) 2 (2) NA Harville et al (520);
Chen et al (518)
Pathogenic/ likely
pathogenic
(VCV000370943)
ARL6 c.123+1131_*25296del53985 53985bp deletion 1 (1) NA Chen et al (518) Not present
(NM_177976.3) c.281T>C p.(Ile94Thr) 1 (7) Remote regions Khan et al (522) Likely pathogenic
c.534A>G p.(Gln178Gln) 1 (2) NA Maria et al (524) Not present
BBS4
NM_033028.5
c.221-1G>A Affects splicing 1 (2) Quetta,
Balochistan
Personal
communication – Dr I
D’Atri
Not present
BBS5
(NM_152384.3)
c.2T>A p.(Met1Lys) 2 (2) NA Harville et al (520) Pathogenic
(VCV000812118)
c.196delA p.(Arg66Glufs*12) 1 (2) Bannu, KPK Khan et al (61) – this
study
Not present
c.734_744del11 p.(Glu245Glyfs*18) 2 (3) NA Maria et al (524) Not present
MKKS
(NM_018848.3)
c.119C>G p.(Ser40*) 1 (3) Nawab Shah
City, Sindh
Ullah et al (a) (528) Likely pathogenic
(VCV000549478)
c.287C>T p.(Ala96Val) 1 (3) NA Ullah et al (b) (527) Not present
260
c.775delA p.(Thr259Leufs*21) 1 (2) NA Ullah et al (b) (527) Pathogenic
(VCV000866319)
BBS7 c.580_582delGCA p.(Ala194del) 1 (3) KPK Ullah et al (a) (528) Not present
(NM_176824.3) c.719G>T p.(Gly240Val) 1 (2) Peshawar, KPK Hayat et al (521) Not present
c.1592_1597delTTCCAG p.(Val531_Pro532del) 1 (3) AJ&K Ullah et al (a) (528) Pathogenic
(VCV000030680)
TTC8 c.235+1G>A Affects splicing 1 (2) NA Deveault et al (519) Not present
(NM_198309.3) c.1019+2_1019+4del Affects splicing 2 (4) NA Harville et al (520);
Ansley et al (80)
Pathogenic
(VCV000002529)
c.1347G>C p.(Gln449His) 1 (3) KPK Ullah et al (a)(528) Pathogenic
(VCV000235131)
BBS9
(NM_198428.3)
c.299delC p.(Ser100Leufs*24) 3 (6) DI Khan, KPK Khan et al (523);
Muzammal et al (525)
Not present
c.1789C>T p.(Gln597*) 1 (1) NA Maria et al (524) Not present
BBS10
(NM_024685.4)
c.271dupT p.(Cys91Leufs*5) 1 (2) AJ&K Ullah et al (a) (528) Pathogenic
(VCV000001328)
c.1075C>T p.(Gln359*) 1 (5) Remote regions Khan et al (522) Not present
c.1958_1967del p.(Ser653Ilefs*4) 1 (2) Punjab Agha et al (514) Not present
BBS12 c.1438delG p.(Arg480Metfs*3) 1 (3) NA Billingsley et al (517) Not present
(NM_152618.3) c.1589T>C p.(Leu530Pro) 2 (2) NA Harville et al (520) Not present
c.2102C>A p.(Ser701*) 1 (3) NA Pawlick et al (526) Not present
Abbreviations: AJ&K, Azad Jammu and Kashmir; BBS, Bardet-Biedl syndrome; KPK, Khyber Pakhtunkhwa; NA, not available. The novel BBS5 variant identified in this study is highlighted in yellow
261
APPENDIX C: GENETIC VARIANTS AND MAPPED LOCI ASSOCIATED WITH NON-
SYNDROMIC AND SYNDROMIC OCA IN PAKISTANI POPULATIONS
Appendix Table C includes a summary of ClinVar entries and PubMed reports defining possible pathogenicity of each variant, as well as other genetic information including the number of families and affected individuals reported
Gene Nucleotide variant
Protein variant References Province (region) [caste]
No of affected
individuals (families)
Variant identified elsewhere
ClinVar (Accession)
TYR c.62C>T p.(Pro21Leu) Jaworek et al (193) Punjab [Sayyid]
1 (15) No Not present
TYR c.103T>C p.(Cys35Arg) Jaworek et al (193) Punjab [Malik; Malik Jutt]
2 (10) No Not present
TYR c.132T>A p.(Ser44Arg) Shaha et al (530); Ullah et al (531);
Arshad et al (532); Unpublished
KPK (Peshawar) 4 (13) No Not present
TYR c.164G>C p.(Cys55Ser) Shahzad et al (186) Punjab [Sidhu jut]
1 (3) No VUS (VCV000617794)
TYR c.223G>T p.(Asp75Tyr) Shahzad et al (186) Punjab [Qureshi]
1 (3) No Likely pathogenic (VCV000617795)
TYR c.230G>A p.(Arg77Gln) Shaha et al (530) AJ&K [Khokar; Rajput]
5 (20) Japanese (201, 223, 226, 533-535); Korean
(536, 537); Chinese (162, 189, 229); Iran
(538); Caucasian (129); North European (131); Scottish (131); Italian (131); German (539); France (153)
Pathogenic/ likely pathogenic
(VCV000003776)
TYR c.240G>C p.(Trp80Cys) Arshad et al (532) KPK 1 (2) No Not present
262
[Pathan]
TYR c.248T>G p.(Val83Gly) Gula et al (188) KPK (Swat) [Pashto]
1 (1) No Not present
TYR c.272G>A p.(Cys91Tyr) Shakil et al (540) Punjab (RYK) [Somro]
1 (4) North American Hutterites (195)
Pathogenic (VCV000039977)
TYR c.308G>A p.(Cys103Tyr) Shakil et al (540) Punjab (Sahiwal) [Khokar]
1 (4) France (141) Not present
TYR c.346C>T p.(Arg116*) Gulb et al (192); Shakil et al (540)
Punjab (Lahore) [Pukhtun; Mughal]
2 (3) Chinese (131, 162, 189, 197, 229); German (539)
Pathogenic (VCV000099565)
TYR c.585G>A p.(Trp195*) Shahzad et al (186) Punjab [Gondal]
1 (6) Indian (541) Likely pathogenic (VCV000617796)
TYR c.593T>C p.(Ile198Thr) Shahb et al (542) NA 1 (4) No Not present
TYR c.649C>T p.(Arg217Trp) Shahzad et al (186); Sajid et al
(191); Arshad et al (532)
Punjab; Sindh [Rajput; Alvi; Mochi; Arain;
Minhas]
8 (41) Caucasian (198) Pathogenic/ likely pathogenic/ VUS (VCV000003795)
TYR c.667C>T p.(Gln223*) Unpublished KPK (Bunar) [Pashton]
1 (2) No Not present
TYR c.715C>T p.(Arg239Trp) Shaha et al (530); Shakil et al (540)
Punjab (RYK) [Turk]
2 (6) Japanese (199); South Indian (200); Chinese
(189)
Not present
TYR c.826T>C p.(Cys276Arg) Bibi et al (205) KPK 1 (3) No Not present
TYR c.832C>T p.(Arg278*) Shahzad et al (186); Jaworek et al
(193); Bibi et al (205); Sajid et al (191);
Forshew et al (543); Arshad et al (532); Shakil et al
(540); Unpublished
Punjab; Sindh; KPK (Lahore;
Peshawar) [Punjabi; Shaikh; Rajput; Mula khel; Arain; Malik; Jutt; Sayyid bukhari;
Bhatti; Urdu-speaking; Gujjar; Mayo; Pashton]
22 (99) Guyana (194); Japanese (201, 226, 227); Chinese (162, 171, 189, 197, 202, 228-230); Korean (231); Indian (131,
215, 223-225); Canada (203); Irish (131); Italian (131);
Mexican (131); South European (131);
Syrian (131); German
Pathogenic (VCV000099583)
263
(131); Polish (131); Israeli
Moroccan/Tunisian Jewish (232)
TYR c.895C>T p.(Arg299Cys) Gula et al (188) Punjab (Ranwal) [Saraiki]
1 (2) Danish (146); Chinese (197);
Not present
TYR c.896G>A p.(Arg299His) Shahzad et al (186); Jaworek et al
(193); Mauri et al (140)
Punjab [Warraich;
Abbassi; Waseer]
4 (15) Caucasian (198); Chinese (189, 197,
230, 544, 545); Israeli Arab Christian (232);
Korean (537)
Pathogenic (VCV000003796)
TYR c.982G>C p.(Glu328Gln) Tripathi et al (194) NA 1 (1) No Not provided (VCV000099591)
TYR c.1037G>T p.(Gly346Val) Sajid et al (191) Sindh 1 (4) Indian (223) Likely pathogenic (VCV000437986)
TYR c.1037G>A p.(Gly346Glu) Shahzad et al (186) Punjab [Sahoo]
1 (3) Chinese (189); Lebanese (546)
Likely pathogenic (VCV000617799)
TYR c.1147G>A p.(Asp383Asn) Shahzad et al (186) Punjab [Bangulzai]
1 (4) Japanese (534); North European (131);
French (131); German (131, 539); Norwegian
(131); Irish (131); Caucasian (134, 547);
Korean (536)
Pathogenic (VCV000003775)
TYR c.1204C>T p.(Arg402*) Shahzad et al (186) Punjab [Gujjar]
1 (5) Israeli Arab Christian (232); East Indian
(215); Chinese (189); France (153); English (131); Scottish (131); German (131, 539);
Italian (131); Lebanese (131, 546); Hungarian
(131); Irish (131); Polish (131);
Caucasian (134, 547)
Pathogenic (VCV000099542)
264
TYR c.1217C>T p.(Pro406Leu) Jaworek et al (193) Punjab [Malik Jutt]
1 (6) France (153); Amish (548); Ashkenazi
Jewish (549); Qatari (550)
Pathogenic (VCV000003777)
TYR c.1231T>C p.(Tyr411His) Jaworek et al (193) Punjab [Arain]
1 (6) No Not present
TYR c.1255G>A p.(Gly419Arg) Shahzad et al (186); Gula et al
(188); Jaworek et al (193);
Gulb et al (192); Tripathi et al (194); Sajid et al (191); King et al (131);
Arshad et al (532); Shakil et al (540);
Unpublished
Punjab; KPK; Sindh; AJ&K
(Chiniot; Gujranwala;
Lahore; Sargodha;
Peshawar; DI Khan; Swat) [Virk; Saraiki; Pashto; Bhatti Jutt; Rajput;
Shaikh; Chanarr; Daaye; Oteera;
Dodiyaanay sial; Sindhi speaking; Urdu speaking;
Saraiki speaking; Chudhar Jutt; Arain; Malik
Awan; Cheema; Pashton]
26 (94) Indian (207, 215, 224); Egyptian (131); German (539);
Caucasian (547)
Pathogenic (VCV000003792)
TYR c.1424G>A p.(Trp475*) Gula et al (188) Punjab (Bhakkar) [Saraiki]
1 (5) No Not present
TYR c.1037-18T>G Affects splicing Shahzad et al (186) Sindh [Urdu-speaking]
1 (4) No Likely pathogenic (VCV000617798)
TYR c.1037-7T>A Affects splicing Shahzad et al (186); Gula et al
(188); Unpublished
Punjab; KPK (Sargodha; DI
Khan)
6 (22) Chinese (189); France (153); Danish (131);
North European (131); Italian (131); Spanish
Pathogenic/ likely pathogenic
(VCV000099527)
265
[Saraiki; Goraya; Sayyid bukhari;
Khokar; Cheema]
(131); German (131); Caucasian (134, 547);
Israeli Moroccan/Sephardic
Jewish (232)
TYR c.1184+2T>C Affects splicing Shahzad et al (186) Punjab (Bhatti) 1 (6) Chinese (551) Likely pathogenic (VCV000617800)
TYR c.344_345del GA
p.(Arg115Thrfs*52) Oetting et al (552) NA 1 (1) German (539); North European (131); Caucasian (547)
Not present
TYR c.943_948del TCAGCT
p.(Ser315_Ala316 delSerAla)
Shahzad et al (186); Forshew et
al (543)
Punjab [Butt]
2 (14) No Likely pathogenic (VCV000617797)
TYR c.1002delA p.(Ala335Leufs*20) Unpublished Punjab (Lahore) [Mayo]
1 (3) No Not present
TYR Exon 4-5 del Frameshift Shahzad et al (186) Punjab [Jutt; Chakotaray]
2 (23) South Indian (Vysya) (224)
-
OCA2 c.827T>A p.(Val276Glu) Sajid et al (191) AJ&K 1 (2) No Not present
OCA2 c.877G>C p.(Glu293Gln) Sajid et al (191) AJ&K 1 (2) No Not present
OCA2 c.954G>A p.(Met318Ile) Jaworek et al (193) Punjab [Warraich]
1 (23) No Not present
OCA2 c.1056A>C p.(Arg352Ser) Shahzad et al (186) Sindh [Sindhi-speaking]
1 (11) No Not present
OCA2 c.1064C>T p.(Ala355Val) Shahzad et al (186) Punjab [Chandia]
1 (5) No Likely pathogenic (VCV000617804)
OCA2 c.1075G>C p.(Gly359Arg) Shahzad et al (186) KPK [Saraiki-speaking]
1 (11) No Likely pathogenic (VCV000452941)
OCA2 c.1211C>T p.(Thr404Met) Shahzad et al (186) Punjab [Sayyid]
1 (5) Nigerian (553) Likely pathogenic (VCV000211766)
OCA2 c.1322A>G p.(Asp441Gly) Shahzad et al (186) Sindh [Sindhi-speaking]
1 (4) No VUS (VCV000502704)
OCA2 c.1327G>A p.(Val443Ile) Mauri et al (140); Arshad et al (532)
Punjab [Saraiki]
2 (2) France (153); North European (212); German (217); Chinese (211)
Pathogenic/ likely pathogenic/ VUS (VCV000000955)
266
OCA2 c.1456G>T p.(Asp486Tyr) Shahzad et al (186); Jaworek et al
(193); Sajid et al (191); Unpublished
Punjab; KPK; Sindh (Qasur;
Borewala) [Lanjay; Mehay; Ghallu; Chaaki; Wains; Bhutta;
Saraiki-speaking; Sindhi-speaking;
Dogar]
12 (61) No VUS (VCV000617806)
OCA2 c.1580T>G p.(Leu527Arg) Jaworek et al (193); Punjab [Warraich]
1 (23) No Not present
OCA2 c.1762C>T p.(Arg588Trp) Arshad et al (532) Punjab [Saraiki]
1 (1) No VUS (VCV000885235)
OCA2 c.1922C>T p.(Ser641Leu) Shahzad et al (186) Punjab [Mughal]
1 (4) No Likely pathogenic (VCV000617807)
OCA2 c.2020C>G p.(Leu674Val) Arshad et al (532) KPK [Yousafzai]
1 (2) East Indian (215) Pathogenic/ VUS (VCV000194918)
OCA2 c.2207C>T p.(Ser736Leu) Unpublished NA 1 (1) Caucasian (208); France (153); China
(209)
Likely pathogenic/ VUS
(VCV000195557)
OCA2 c.2228C>T p.(Pro743Leu) Shahzad et al (186); Jaworek et al
(193)
Punjab; Sindh [Arain; Joyia;
Urdu-speaking]
3 (19) North European (212); Chinese (189)
Pathogenic/ likely pathogenic
(VCV000000956)
OCA2 c.2359G>A p.(Ala787Thr) Jaworek et al (193) Punjab [Chohan]
1 (4) France (153); Chinese (554)
Pathogenic (VCV000803060)
OCA2 c.2360C>T p.(Ala787Val) Shahzad et al (186); Unpublished
KPK, Punjab (Bajaur) [Arain]
2 (6) Yes (210) Pathogenic/ likely pathogenic
(VCV000617810)
OCA2 c.2360C>A p.(Ala787Glu) Shahzad et al (186) Punjab [Langah Jutt]
1 (9) No Likely pathogenic (VCV000617809)
OCA2 c.2458T>C p.(Ser820Pro) Arshad et al (532) Punjab [Niaz]
1 (2) No Not present
OCA2 c.1045-15T>G Affects splicing Shahzad et al (186); Jaworek et al
Punjab; KPK; Sindh (Peshawar)
[Niaz; Afridi;
18 (70) No Likely pathogenic (VCV000617802)
267
(193); Arshad et al (532); Unpublished
Bubar; Sidhu Jutt; Abbassi; Ansari;
Rajput; Jutt; Arain, Bhatti;
Sindhi-speaking; Saraiki-speaking;
Pashton]
OCA2 c.1182+2dupT Affects splicing Shahzad et al (186) Punjab [Arain]
1 (3) No Likely pathogenic (VCV000617805)
OCA2 c.1951+4A>G Affects splicing Shahzad et al (186) Sindh [Urdu-speaking]
1 (5) No Likely pathogenic (VCV000617808)
OCA2 c.408_409del AA
p.(Arg137Ilefs*83) Arshad et al (532); Unpublished
KPK (Peshawar) [Yousafzai; Pashton]
2 (4) No Not present
OCA2 c.987delCins AGA
p.(Gln330Aspfs*2) Shahzad et al (186) Sindh 1 (5) No Likely pathogenic (VCV000617801)
OCA2 c.1960delG p.(Ala654Leufs*8) Lee et al (212) NA 1 (3) No Pathogenic (VCV000000957)
OCA2 Exon 3-14 del Frameshift Shahzad et al (186) Punjab [Machi]
1 (3) No -
OCA2 Exon 7-8 del Frameshift Shahzad et al (186); Mauri et al
(140)
Punjab [Mughal; Terkhan]
2 (8) No -
OCA2 Exon 19 del Frameshift Shahzad et al (186); Gula et al
(188); Unpublished
Punjab (Tank) [Pashto; Malik]
4 (18) No -
OCA2 Exon 20-24 del
Frameshift Shahzad et al (186) Punjab [Achakzai]
1 (5) No -
OCA2 Exon 22-24 del
Frameshift Shahzad et al (186) Punjab [Rajput]
1 (3) No -
TYRP1 c.256G>T p.(Asp86Tyr) Kausara et al (555) Punjab [Bhutta]
1 (3) No Likely pathogenic (VCV000617811)
TYRP1 c.1067G>A p.(Arg356Gln) Shahzad et al (186) Punjab [Langah; Haraaj;
Matam]
3 (12) Caucasian (556), Chinese (557)
Pathogenic/ likely pathogenic
(VCV000017596)
268
TYRP1 c.1120C>T p.(Arg374*) Forshew et al (543) NA 1 (8) No Pathogenic (VCV000017595)
TYRP1 c.1534C>T p.(Glu512*) Shahzad et al (186) Punjab [Shaikh]
1 (7) No Likely pathogenic (VCV000617812)
TYRP1 c.647_668del p.(Glu216Glyfs*42) Kausara et al (555) Punjab [Ladd]
1 (5) No Not present
TYRP1 ~1 Mb del including whole gene Gula et al (188) KPK (Lakki Marwat) [Pashto]
1 (2) No -
SLC45A2 c.251T>C p.(Leu84Pro) Kausara et al (555) Punjab [Lakhat]
1 (4) No Not present
SLC45A2 c.1532C>T p.(Ala511Val) Shahzad et al (186); Gula et al
(188); Kausara et al (555)
Punjab; KPK (Kech) [Saraiki;
Saraiki-speaking; Kingray]
4 (17) No Likely pathogenic (VCV000617813)
SLC45A2 c.889-6T>G Affects splicing Kausara et al (555) Punjab [Rehmani]
1 (3) No Not present
SLC45A2 c.1331_dupA p.(Asn444Lysfs*5) Bibi et al (205) KPK 1 (2) No Not present
OCA5 Mapped locus Kausarb et al (117) Punjab 1 (6) No -
MC1R c.917G>A p.(Arg306His) Saleha et al (558) NA 1 (3) No VUS/ benign (VCV000470716)
LYST c.9827_9832 delATACAA
p.(Asn3276_Thr3277del)
Weisfeld-Adams et al (559)
NA 1 (3) No Pathogenic (VCV000180629)
HPS1 c.437G>A p.(Trp146*) Unpublished Punjab (Shadra) [Bhatti Rajpoot]
1 (2) No Not present
HPS1 c.517C>T p.(Arg173*) Unpublished KPK (Swabi) [Pashton, Yousafzai]
1 (1) Chinese (402) Pathogenic (VCV000937794)
HPS1 c.1342T>C p.(Trp448Arg) Yousaf et al (560) Punjab 1 (5) No Likely pathogenic (VCV000690341)
HPS1 c.2009T>C p.(Leu670Pro) Unpublished KPK (Peshawar) [Pashton]
1 (3) No Not present
HPS1 c.2056C>T p.(Gln686*) Yousaf et al (560) Punjab 1 (4) No Likely pathogenic (VCV000690342)
269
HPS1 c.1397+1G>A Affects splicing Unpublished Balochistan [Baloch]
1 (1) No Not present
HPS1 c.972delC p.(Met325Trpfs*6) Unpublished Puunjab (RYK) [Bukhari Sayyed]
1 (3) Japanese (401) Pathogenic (VCV000005280)
HPS1 c.118-105_133 del121
p.(Leu40Thrfs*7) Yousaf et al (560) Punjab 1 (5) No -
HPS3 c.1509G>A p.(Met503IIe) Yousaf et al (560) Punjab 1 (4) No Likely pathogenic (VCV000627005)
HPS4 c.276+5G>A Affects splicing Yousaf et al (560) Sindh 1 (5) No Not present
HPS6 c.823C>T p.(Pro275Ser) Yousaf et al (560) Punjab 1 (5) No Likely pathogenic (VCV000690344)
BLOC1S3 c.448delC p.(Gln150Argfs*75) Morgan et al (561) NA 1 (6) No Pathogenic (VCV000001489)
BLOC1S6 c.232C>T p.(Gln78*) Yousaf et al (560) Sindh 1 (2) North Italian (562) Pathogenic (VCV000030412)
Abbreviations: AJ&K, Azad Jammu and Kashmir; del, deletion; KPK, Khyber Pakhtunkhwa; NA, not available; RYK, Rahim Yar Khan; VUS, variant of uncertain significance. Study families are highlighted in red.
270
APPENDIX D: PRIMER PAIRS AND PCR CONDITIONS
Table D1 Primer pairs used for sequencing the TYR coding exons and
associated intron-exon junctions
Primer Primer Sequence 5’ → 3’ Annealing
temp (°C)
Amplicon
size (bp)
TYR_Exon 1_F TCAGCCAAGACATGTGATAATCA 60 992
TYR_Exon 1_R TTATACCCTGCCTGAAGAAGTG
TYR_Exon 2_F CAACATTTCTGCCTTCTCCTA 55 888
TYR_Exon 2_R CTGCCTAGAATATTTTAAACAGG
TYR_Exon 3_F GAATGAACAGGAGGGAACAC 58 470
TYR_Exon 3_R TCTATTTAAATCCAATGAGCACGTT
TYR_Exon 4_F TTCTGGAGGTTCAAAACTCAATG 58 675
TYR_Exon 4_R ACAAAATGGCCTATGTTAAGCAA
TYR_Exon 5_F TGTCTACTCCAAAGGACTGT 54 921
TYR_Exon 5_R GGCACTTAGCTGGATGTGTT
Table D2 Primer pairs used for sequencing the inherited eye disease gene
variants identified in the study
Primer Primer Sequence 5’ → 3’ Annealing
temp (°C)
Amplicon
size (bp)
ALDH1A3_G414R_F CAGTCTCCAATGGCAATGCA 60 359
ALDH1A3_R414R_R CACACACAGTCAACTCACCA
ALDH1A3_c172dupG_F GGTGGACAAGATGGATAAGACG 58 300
ALDH1A3_c172dupG_R GGCCAGTTCTGTCTTATAGCT
ATOH7_c94delG_F GGAAGCCGAAGAGTCTCTGG 57 592
ATOH7_c94delG_F GCACTCCCCCACTGTAAACT
BBS1_M390R_F TTCCCCAGGCCTGTCTCTAT 60 539
BBS1_M390R_R TCCGTCTTCCAGACGAGACT
BBS5_ c196delA_F ACACATATGACTTGCTGGGAC 56 676
BBS5_ c196delA_R TGGGATTAATCAAAACAGGGGA
CYP1B1_R390H_F TCATCACTCTGCTGGTCAGG 58 388
CYP1B1_R390H_R GAATTTTGCTCACTTGCTTTTC
FRMD7_L148*_F ATCTCAGCGTTTCATGGAGC 57 237
271
FRMD7_L148*_R TGCAGCAGAACTTGGAGACT
FYCO1_Q736*_F CTTAGCTGGCTCTGCACCTT 58 363
FYCO1_Q736*_R CCAGATGGCAGAGAAGAAGG
HPS1_W146*_F TGCTTGTGCCTTCATTCATT 56 388
HPS1_W146*_R CCCCACTCCACAGTTAGAGC
HPS1_R173*_F AGAGTAGAATGCCAGCAGCTT 64 243
HPS1_R173*_R AGTGAATGTCCCCACTAGCC
HPS1_L670P_F ACAGGATGCAAAGGCAGACT 60 240
HPS1_L670P_R CCACAGCCTCACTCCTGAAT
HPS1_c972delC_F ACAATGGAGCTGAGGGACAG 58 468
HPS1_c972delC_R TTAGGATGAAGGGGTGTTGC
HPS1_c1391+1_F GGGCATTACAGCAGAAGGGA 61 435
HPS1_c1391+1_R CAAAAGTGAGCCCGGATCCT
INPP5E_ Q627*_F AGGGTTGGCTTCCTTCCTG 58 392
INPP5E_ Q627*_R GCCCTGGGTGTCCTCTTAAA
LRP5_T359R_F GCCTGGCTGAGTATTTCCCT 58 580
LRP5_T359R_R CCAGAATGACAGGTCCAGGT
MKKS_H84Y_F TCGACAACCACAGGTCTCAG 60 240
MKKS_H84Y_R TCCTCAGCTCTGCTCAGTCA
OCA2_ V443I_F GGACTGGAATGCAGTGAGCT 64 321
OCA2_ V443I_R TCTACGAGCCTGCTCACTCT
OCA2_ D486Y_F CTTCCTCAGCTCTTGGTTGG 60 230
OCA2_ D486Y_R ATACGAGCAAGCGCCTTAAA
OCA2_ R588W_F TGGTTTTTGTCGTGCAACAA 64 578
OCA2_ R588W_R CATCGACTGTGTGGGGAACA
OCA2_ L674V_F ACAAATACGCAGTGCTGTCAG 62 320
OCA2_ L674V_R TTCTGGGTGCCATCTGGTTG
OCA2_ S736L_F TACACCTGTGAGTGCAGCAG 56 567
OCA2_ S736L_R GTAAATGAGTGCTGCAGGCG
OCA2_ Ala787V_F CCTGAAAAATTCCATGAAGGAG 58 196
OCA2_ Ala787V_R CAAATCAAAGCCTGTGAGATGA
OCA2_ S820P_F TCTGGAGGGGAATCTTGAGT 56 551
OCA2_ S820P_R TGCACACAATGGAGGATGTC
OCA2_c1045-15_F AGTTCTGTGCACGATCTGGA 58 184
OCA2_c1045-15_R TATGTGTCTGTGGGGTGTCC
OCA2_c408delAA_F GTCTCCCTAGGCCCAGGTAA 62 501
OCA2_c408delAA_R AGAGTGCACCACTGTCTGTG
PAX6_R240*_F TGAATCACAAAGTGTGAAACTGC 64 206
PAX6_R240*_R AGGTGGGAACCAGTTTGATG
272
SCAPER_c2236dup_F TTTGATCTCATGCTCCACTGT 56 401
SCAPER_c2236dup_R AGCCCTTTAGACTATATGACCCT
SDHD_E69K_F CTGCCTGTCAGTTTGGGTTA 58 243
SDHD_E69K_R TTGCCAGTGACCATGAAGAG
TDRD7_c2469delG_F GGATTTTAGCAAGGGTTTTGG 60 396
TDRD7_c2469delG_R GAGAAAGCGCTCGTATGGTC
273
APPENDIX E: PUBLICATIONS RELATING TO THIS
RESEARCH
First/joint first author Lin S, Haralka GV, Hameed A, Reham HM, Yasin M, Muhammad N, Khan S, Baple EL, Crosby AH, Saleha S. Novel mutations in ALDH1A3 associated with autosomal recessive anophthalmia/microphthalmia, and a review of literature. BMC Medical Genetics 2018 Sep 10; 19 (1):160 Lin S, Sanchez-Bretaño A, Leslie JS, Williams KB, Lee H, Thomas NS, Callaway J, Deline J, Ratnayaka JA, Barelle D, Schmitt MA, Norman CS, Hammond S, Harlalka GV, Ennis S, Cross HE, Crosby AH, Baple EL, Self JE. Evidence that the Ser192Tyr/Arg402Gln in cis Tyrosinase gene haplotype is a disease-causing allele in oculocutaneous albinism type 1B (OCA1B). In submission to npj Genomic Medicine Lin S*, Fasham J*, Al-Hijawi F*, Qutob N, Gunning A, Leslie JL, McGavin L, Ubeyratna N, Baker W, Zeid R, Turnpenny PD, Crosby AH, Baple EL, Khalaf-Nazzal R. Consolidating biallelic SDHD variants as a cause of mitochondrial complex II deficiency. Eur J Hum Genet 2021 May 20. doi: 10.1038/s41431-021-00887-w. Online ahead of print Fasham J*, Arno G*, Lin S*, Xu M, Carss KJ, Hull S, Lane A, Robson AG, NIHR Bioresource Rare Diseases Consortium, Wenger O, Self JE, Harlalka GV, Salter CG, Schema L, Moss TJ, Cheetham ME, Moore AT, Raymond FL, Chen R, Baple EL, Webster AR, Crosby AH; NIHR Bioresource Rare Diseases Consortium. Delineating the expanding phenotype associated with SCAPER gene mutation. Am J Med Genet A 2019 Aug; 179(8):1655-1671 Co-author
Shakil M, Harlalka GV, Ali S, Lin S, D’Atri I, Hussain S, Nasir A, Shahzad M, Ullah M, Self JE, Baple E, Crosby A, Mahmood S. Tyrosinase (TYR) gene sequencing and literature review reveals recurrent mutations and multiple founder gene mutations as causative of oculocutaneous albinism (OCA) in Pakistani families. Eye (Lond) 2019 Aug; 33(8): 1339-1346 Khan S, Lin S, Harlalka GV, Ullah A, Shah K, Khalid S, Mehmood S, Hassan MJ, Ahmad W, Self JE, Crosby AH, Baple EL, Gul A. BBS5 and INPP5E mutations associated with ciliopathy disorders in families from Pakistan. Ann Hum Genet 2019 Nov; 83(6): 477-482 Arshad MW, Harlalka GV, Lin S, D’Atri I, Mehmood S, Shakil M, Hassan MJ, Chioza BA, Self JE, Ennis S, O’Gorman L, Norman C, Aman T, Ali SS, Kaul H, Baple EL, Crosby AH, Ullah MI, Shabbir MI. Mutations in TYR and OCA2 associated with oculocutaneous albinism in Pakistani families. Meta Gene 2018 Sept; 17: 48-55